90a Membrane Domains and Polarity (520-522). Sunday

520 521 CHOLESTEROL VARIATION IN COCHLEAR OUTER HAIR CELL TRANSMEMBRANE LIPID ASYMMETRY AND FLIP-FLOP IN MEMBRANE DOMAINS DIABETIC ERYTHROCYTES ((Michael J. Wilson, Eric B. Sputh, Jason Cholesterol is an important membrane constituent that influences B. Butler, Delaina Eash, David L. Daleke.)) Medical Sciences Program, membrane fluidity and the activity of membrane-bound enzymes. Indiana University, Bloomington, IN 47405 Cholesterol is not uniformly distributed within the cochlear outer hair cell Exposure of non-diabetic erythrocytes to hyperglycemic conditions in vitro membrane. Functionally, this cell can be divided into three domains: the alters transmembrane phospholipid asymmetry, induces the expression of apex plays a role in mechanico-electrical transduction; the base is involved phosphatidylserine (PS) on the cell surface, and converts the cells to a pro- with synaptic transmission; and the lateral wall exerts electromotility, or coagulant state. Loss of asymmetry is accompanied by an increase in lipid rapid length changes in response to electrical energy. Using filipin, a oxidation and depletion of cellular o-tocopherol and can be suppressed with polyene fluorescent antibiotic that binds to cholesterol, we find that the a combination of lipophilic and hydrophilic antioxidants. Additional in vitro lateral wall contains less cholesterol than the apical and basal membranes. experiments have demonstrated that the likely mechanism is an increase in This difference in filipin fluorescence between the lateral walls and the ends passive lipid flip-flop; bidirectional movements of short chain analogs of each diminishes when cells are incubated in water-soluble cholesterol prior to class of endogenous erythrocyte phospholipid are increased 2-20 fold in cells staining, suggesting that exogenous cholesterol preferentially enters the treated with high concentrations of glucose. These results suggest that hyper- confocal we the lateral wall. Under microscopy, study incorporation pattern glycemia induces an oxidant-mediated increase in passive lipid flip-flop which a of fluorescent cholesterol analogue, NBD-cholesterol. The confocal results in an apparent loss of asymmetry. In the present work, we examine findings are consistent with the filipin results in that NBD-cholesterol does the oxidative status of, and transmembrane lipid movements in, erythrocytes while it labels the lateral wall. The not stain the apical membrane intensely isolated from diabetic individuals and rats. Lipid oxidation by-products were micropipette aspiration technique is used to assess the effect of cholesterol increased in diabetic erythrocytes but the activities of catalase and glutathione on lateral wall stiffness. The stiffness cholesterol-treated = of cells (n 23) is peroxidase were unchanged. Passive lipid flip-flop of di[C10:0]PC and [1-"C]- than that of controls = = 0.76 0.24 significantly higher (n 27): S + (mean 1-palmitoyl-lyso PC was increased in erythrocytes from diabetic animals and versus S = 0.46 + Student's = 5 E-6. In SD) 0.10, t-test, p conclusion, poorly-controlled diabetic humans, but not in well-controlled diabetics or an- cholesterol has different distributions among the three membrane domains, imals treated with insulin to normalize plasma glucose content. Furthermore, and when an additional amount is incorporated, cholesterol will decrease the erythrocytes from poorly controlled diabetic individuals displayed increased elasticity of the outer hair cell, which may affect electromotility. This project is supported by the American Academy of prothrombin converting activity than cells from well-controlled individuals. These results support a model in which glucose induces an oxidant-mediated Otolaryngology -- Head and Neck Surgery Foundation Resident Research Grant and the NIDCD/DC-00354 Grant. increase in transbilayer lipid movements that converts cells to a procoagulant state. These perturbations may contribute to the microvascular complications of the disease.

522 DETERGENT-RESISTANT, GLYCOSPHINGOLIPID-ENRICHED, MEMBRANOUS MICRODOMAINS CONTAIN BOTH THE CELLULAR AND SCRAPIE PRION PROTEINS. Martin Vey', Susanne Pilkuhnl, Holger Willel, Richard G. W. Anderson2, and Stanley B. Prusinerl. 1University of California, San Francisco, CA 94143; 2University of Texas Southwestern Medical Center, Dallas, TX 75235. GPI-anchored proteins cluster in detergent-resistant membranous microdomains called DIGs (detergent-insoluble, glycosphingolipid- enriched complexes) which are enriched for both cholesterol and glycosphingolipids. To determine if prion protein (PrP) also associates with these membrane domains, we used two independent methods for isolation of DIGs from scrapie-infected and uninfected mouse neuroblastoma cells. In the detergent-based method, cells were lysed in cold Triton-X100 and the DIGs were separated from the lysates by flotation in sucrose gradients. Alternatively, postnuclear supernatants were prepared from cell homogenates in the absence of detergent and fractionated on Percoll gradients. Isolated plasma membrane fractions were further disrupted by sonication and the DIGs separated from membranous debris by flotation in OptiPrep gradients. Analysis of DIGs showed that the cellular prion protein (PrPc)was concentrated in DIGs. Prion infection did not interfere with formation of DIGs and even after conversion, the pathologic, scrapie isoform PrPsc was still associated with them. The PrP 27-30 core of PrPSc was detected after limited digestion with proteinase K. Furthermore, a 17 kDa degradation product of PrPC could be identified. These results raise the possibility that prion propagation occurs in microdomains of cellular membranes which are enriched for cholesterol, glycosphingolipids and GPI-anchored proteins. GapJunctions I (523-524).

523 524 FREEZE-FRACTURE ANALYSIS OF GAP JUNCTIONS AND SQUARE ARRAYS AT DOCKING SPECIFICITY BETWEEN CONNEXINS IS MODIFIED BY THIE SUB-NANOMETER RESOLUTION. ((J. Rash and T. Yasumura)), Department of TERTIARY STRUCTURE OF THE EXTRACELLULAR LOOPS, INDEPENDENT Anatomy and Neurobiology, Colorado State University, Ft. Collins, CO 80523 OF PRIMARY SEQUENCE. ((Brnce J. Nicholson, Xing Zhu, Hui Zhu and Cynthia I. Foote)). Dept Biological Sciences, SUNY at Buffalo, Buffalo, NY 14260 A new method for deposition of continuous, subnanometer-thick platinum/carbon ((Jurgen films on freeze-fractured membranes from rat liver and spinal cord reveals structural Schwarz and Klaus Willecke)). Inst. for Genetik, U. Bonn., Germany. details smaller than 1 nm. Stereoscopic Images of gap junction P-faces reveal connexons as conical tubes 6-7 nm In diameter, each containing a central 2 nm The specificity of docking between liemichannels composed of different connexins dimple' at Its apex. The corresponding E-face pits are 7-8 nm In diameter, with to form an intact heterotypic gap junction is dictated by the two extracellular loops Intervening lipidic septa forming a continuous honeycomb of 2 nm-thick ridges, all of (El & E2). To achieve a better understanding of the docking process, we recently which fracture at the same height as the extrajunctbonal E-face lipids. Within each undertook systesadic movements ofthe 6 conseved cysteines within the extcellular E-face pit, a central 'peg' corresponding to the aqueous matrix of the connexon Ion loops (3 in ea) in order to map the pattern of disulfide bridges (all disulfides are channel (J. Coll Blol. 115: 190a, 1991) Is resolved. At 600 local shadowing angle, known to form within a single connexin). All single movements predictably produced each peg exhibits a 1.3 nm cap of platinum, whereas, at 90° shadowing angie, the non-functional channels whenpairedwidi wtCx32. Th pattenofpairedmiant diat pegs are resolved as cones with a basal diameter of 3 nm and projecting from a pit rescue coupling demonstrated that all 3 disulfides form between the loops that appear whose bottom has been reduced to 4 nm In diameter. Slmilar-quality replicas from to be in P-sheet confirmation based on the periodicity ofmovements. Among the non- spinal cord astrocytes reveal new details In E-face Images of square arrays". Unlike functional mutants, two revealed a surprising result (one single mutant and one In gap lipic Images square arrays those junctions, the septa within E-face of fracture inappropriately paired mutant). While these failed to pair with wtCx32, they now 1.5-2 nm below the surrounding fracture face. The E-face of square arrays are pits formed hoterotypic channels with XenCx38, a pairing which does not occur in the 5 nm In diameter, with each pit containing a smame, conical peg. At 600 local case of wtCx32. Thus, a change in the folding patten of these regions with minimal shadowing angle, the tip of each peg has a 0.7 nm diameter cap of platinum, which change the primary sequence causes a marked suppression of docking characteristics is one-half the diameter of the pegs in adjacent gap junctions. Stereoscopic Imaging and a concommitant modification ofvoltage gating. In independent experiments, we reveals that the two previousiy-identified morphologies of square arrays result examined docking specificity using a chimeric construct of Cx40 with El and E2 primarily from two primary patterns of deposition of platinum on diagonally- vs contributed by Cx43. wtCx40 does not form heterotypic junctions with either Cx43 perpendicularly-oriented rows of pits in the orthogonal lattices of 'square arrays". or As These data show that very thin (< 1 nm) continuous films yield freeze-fracture replicas XenC38. expected, the chimera no longer couples with wt Cx40. Surprisingly, having a 2-4 fold improvement In resolution over those obtained by previous while it does now couple with XenCx38 (a property of wtCx43X the chimera fails to shadowing methods. Similar levels of detail are resolvable In replicas of other classes couple with wtCx43. This result also suggests that the primary sequence alone does of membrane receptors, pumps, and channels. not dictate docking specificity, but that this is influenced by the tertiary stnucture of This research was supported by NIH grant #RO1 NS-31027. the loops dictated by the disposition of the transmembrane helices. Sunday. Gap Junctions I (525-530) 91a

525 526 DIRECT MEASUREMENT OF SELECTIVE SPECIFICATION OF GAP JUNCTION CHANNEL SUBUNIT ASSEMBLY. PERMEABILITY AMONG UNCHARGED TRACERS AND CYCLIC ((M. M. Falk, L. K. Buehler, N. M. Kumar, and N. B. Gilula)) NUCLEOTIDES BY RECONSTITUTED CONNEXIN CHANNELS ((C.G. Bevans & Cell Biology, The Scripps Research Institute, La Jolla, A.L. Harris)) Biophysics Department, Johns Hopkins University, Baltimore, MD 21218 Gap junction (GJ) channels are complex oligomeric plasma tures, in which six homologous polypeptide subunits known A connexin-specific difference in permeant size cut-off among uncharged tracer recognize each other and assemble in a non-covalent interaction molecules and cyclic nucleotides was positively identified for connexin (Cx) ous pore, similar to ion channels. Two of these structurally channels in a liposome system. Permeability was assessed by comparison of the termed connexons, each provided by one of two adjacent cells, pair tracers retained by liposomes containing purified, functional Cx channels with the complete, double-membrane intercellular junction that functions tracers retained by liposomes not containing functional channels. The uncharged 13 different Cx subunit isotypes are known cell communication. rodents, tracers (a mixture of tri- through hexa-saccharides labeled with an uncharged and different combinations are coexpressed in various cell fluorescent amino-pyridyl group) were recovered from the two populations of allowing the assembly of many different homomeric and heteromeric liposomes, separated by HPLC, and quantitated. Selective loss/retention of subtypes. To investigate GJ channel subunit assembly, we expressed tracers in the liposome populations containing functional/non-functional channels Cx isotypes (al, in translation-competent cell lysates supplemented pj, 02) defined a permeant size cut-off, independent of any charge selectivity. microsomal membranes. SDS-PAGE analysis, characterization Homomeric Cx32 channels (from rat liver, B/ochem. 35:9212) were permeable to topology (N-glycosylation site tagging, protease protection assays), the trisaccharide tracer, but not to the larger tracers. Heteromeric Cx32/Cx26 namic analysis (sedimentation on linear 5 20 % sucrose gradients), channels (from mouse liver) were impermeable to all the sugar tracers, physiological characterization (single channel activity recorded suggesting that Cx26 contributes to a narrowed pore. The size discriminations containing cell-free expressed Cxs) showed that membrane integrated, occur over a range that suggests they are significant in determining connexin- folded Cxs were synthesized that assembled into functional, nonfunctional specific selectivity among biological signaling molecules. This was further channels (connexons). Immuno-coprecipitation experiments al, investigated by studies in which radiolabled cAMP and cGMP were used as the and Cx isotypes indicated that the GJ channel subunits P, tracers. Homomeric Cx32 channels were permeable to both nucleotides, whereas each other and discriminate between different nize selectively only a small fraction of the heteromeric Cx32/Cx26 channels were permeable to assemble only into homomeric, and into certain permitted heteromeric them. This supports the idea that Cx26 contributes to a narrowed pore. It further subtypes. Furthermore, cotranslation of truncated Cx polypeptides suggests that the heteromeric channel population is heterogeneous with regard to this selective subunit assembly is critically driven by selectivity and therefore composed of Cx32 and Cx26 in different stoichiometries segments in the N-terminal portion (N-terminal domain and first and/or arrangements. These findings suggest that intercellular to domain) of the Cx polypeptides. Comparable data have been permeability specific cyclic nucleotides may be regulated by the connexin composition of channels, and recently, heteromeric Cx assembly has also been observed junctional channels. Support: NIH GM36044 in vivo studies. (Supported by NIH, Lucille. P. Markey Charitable Trust, and

527 528 THE ROLE OF RESIDUES ON PH GAP JUNCTION (GJ) HEMICHANNEL ANALYSIS IN HELA HISTIDINE SENSITIVITY OF THE GAP *M. 43. TRANSFECTED WITH DIFFERENT CONNEXINS. ((*H. Li, JUNCTION PROTEIN CONNEXIN ((M.M.P. Hermans, P. Kortekaas, H.J. Atkinson, #C. Elfgang, #K. Willecke, *R. G. Johnson))*Dept. of Jongsma and M.B. Rook)) Department of Medical Physiology and Sports Medicine, and Cell Biology, Univ. of Minnesota, St. Paul, MN 55108, #Inst. University of Utrecht, P.O. Box 80043, 3508 TA Utrecht, The Netherlands. Genetics, Univ.of Bonn, Bonn, Germany. Protonation of histidine residues in the cytoplasmic Wepreviously reported that cells take up fluorescent dyes from loop of connexins has been a proposed to play important role in pH via discrete pathway, in the presence of reduced external calcium (Li an regulation of gap junctional permeability 1996. J. CellBiol., in press).Since the pathway displayedproperties in heart. To study the role on pH gating of histidines (H) 126 and 142 in connexin43 to and since dye uptake correlated withthe expression GJ channels (Cx43), the major cardiac gap junction protein, these residues were substituted by was that Cx43 formed GJ hemichannels connexin43 (Cx43), the interpretation glutamine (Q) via site directed mutagenesis. Wild type and mutant Cx43 cDNA's in the plasma membrane. To determine whether other connexins are were stably transfected into coupling-deficient SkHepl cells. Junctional conductance form comparable pathways, dye uptake was evaluated in was the dual transfected with Cx26, 31, 32,37,4, 43, and 45 (Elfgang et al., (ga) between cell pairs measured using voltage clamp technique, intracellular pH (p11 ) was manipulated with the NH3INH4 Cell Biol., 129: 805-817). All transfectants displayed dye uptake pH-clamp technique. Under normal conditions (pH, 7.0), in the transfected cells was increased sharply with the reduction in external calcium. Quantitative analyses g, substantially higher of both dye uptake and dye transfer in all the transfectants revealed than in parental cells (up to 50 nS versus 3 nS). At a pH, of 6.3,gj in cells expressing extent of dye uptake generally correlated with the GJ permeance wild type Cx43 gap junctions was reduced by 55% of control values, in Cx43-H126Q given population of transfectants. 18-a-glycyrrhetinic acid, a reagent cells this reduction was only 25%, while in Cx43-H142Q cellsg. dropped to zero. to inhibited dye uptake in all transfectants. reduce GJpermeability, For Cx43-H126/142Q cells the results were variable: the reduction in g, ranged compare the regulation of the hemichannels with that of the corresponding between 20 and 80%. At pH, 5.8, most transfectants became completely uncoupled, channels, the effects of the protein kinase C activator, TPA-P, were with the exception of Cx43-H126/142Q cells, where the change ing, ranged between TPA-P, but not TPA-a (the inactive isoform), markedly inhibited dye uptake 0 and 40%. Any pH induced uncoupling was reversible upon returning to 7.0. all but the Cx26 transfectants. This widespread response to TPA suggests pH, At g, levels approaching zero, single channel events could PKC generally serves as a negative regulator of hemichannels. The be recorded. Single TPA-5 on GJ was more channel conductances (y1) of Cx43 wild type and permeance variable, with the most extensive mutant channels were similar: -40 1 same at pH, after 60 minutes observed with Cx3 and 32 and no effect detected 50pS. The y, could be measured 7.0 when the cells were uncoupled effects on other transfectants were more difficult to assess and with halothane. From our data we conclude that the pH sensitivity of Cx43 is study, as are the effects of 8Br-cAMP on uptake and permeance. influenced by the position rather than by the total number of histidine residues in its existenceof plasma membrane hemichannels provides further support for the cytoplasmic loop. pH induced uncoupling results in a reduction of gap junction and indicates that the presence of these channels in GJ-competent cells channel open probability but not in channel conductance. function of connexin type.

530 529 EFFECTS OF PH ON CX46 HEMICHANNELS ((ERB. Trexler, M.V.L. CHARACTERIZATION OF THE pHi SENSITIVITY OF CONNEXIN Bennett, Bargiello, and V.K. Verselis.)) Department of GAP JUNCTION CHANNELS. ((J.F. Ek-Vitorin, N. Homma, G. Calero T.A. Neuroscience, Albert Einstein College of Medicine, Bronx NY 10461 S.M. Taffeta and M. Delmar)) Departments of Pharmacology and Increased levels of cytoplasmic H+ ions have been shown to rapidly, and of- Microbiology and Immunology* SUNY Health Science Center, Syracuse NY 13210. ten reversibly, decrease gap junctional conductance, gi, in a variety of tissues. Unlike decreases in produced by transjunctional voltage,Vj,the uncoupling leads to gap junction channel closure. zero. Intracellular acidification is complete, i.e., gi is reduced to Whether H+ acts directly on connexins susceptibility to acidification-induced uncoupling varies among connexins. remains in question, and the mechanism and site(s) of action are unknown. seem highly susceptible to closure at particular, Cx46 channels to be To address these questions we utilized the ability of Cx46 to function as a et 125:879-892, 1994). However, a quantitative (White al. J Cell Biol hemichannel in isolation, i.e., without an opposed hemichannel. Membrane analysis of the pHi dependence of Cx46 is yet to be We conducted. currents induced by depolarization of single Xenopts oocytes expressing Cx46 techniques to correlate pHi with the junctional optical and electrophysiological and junctional currents in oocyte pairs expressing Cx46 were highly sensitive conductance (Gj) of Cx46 and of other connexin channels. to cytoplasmic acidification. When patches containing single Cx46 hemichan- expressed in Xenopus oocyte pairs, previously injected with Cx38 nels were excised in an inside-out configuration, sensitivity to H+ remained Gj was electrophysiologically. pHi was measured from determined even in the absence of Ca2+. The action of H+ resulted in complete closure emission of the proton sensitive fluorophore dextran-SNARF. of the channel at voltages where transitions to the closed state were rare. Re- acidified by progressively increasing the concentration Of CO2 ductions in single channel conductance were also evident en route so closure superfusate. We found that the Gj of Cx46 channels was modified suggesting there may be a fast blocking component. Some patches excised af- even within normal ranges (Cx46: pKa= 7.07±0.05; Hill coefficient= ter repetitive cycles of acidification applied to the oocyte showed insensitivity 1.84±0.11; Mean±SEM; n=6); Gj through endogenous Cx38 to H+. Taken together with the observations that His residues in the cyto- (paired oocytes not injected with antisense) was also highly dependent plasmic loop of Cx43 significantly affectH+ sensitivity (Ek et al., 1994), we (Cx38: pKa= 7.15±0.02; Hill coefficient= 6.7 ±0.55; n=6). Both propose that that H+ acts directly on the cytoplasmic aspect of the channel, were more sensitive than Cx43, and far more sensitive than Cx32 (see hut that secondary channel modifications, perhaps involving phosphorylation, et al. J As initially by Hermans et al Biophys 70:1294-1302, 1996). proposed strongly influence the action of H+. (Pflugers Arch. Eur J Physiol. 431:138-140, 1995), Cx45 was susceptible to acidification-induced uncoupling than Cx43, though sensitive as Cx38 or Cx46. These results demonstrate that differences primary sequence of connexin channels lead to important disparities junction regulation. pHi may be a rapid, and effective way to fine-tune degree of communication between cells expressing specific connexins. 92a GapJunctions I (531-536). Sunday

531 532 GAP JUNCTION-MEDIATED INTERCELLULAR CALCIUM WAVES IN RAT EFFECT OF CHANGING EXTERNAL CALCIUM ON RAT CX46 AND INSULINOMA CELLS BY ACTIVATION OF VOLTAGE-GATED CALCIUM CHICK CX56 HEMI-GAP JUNCTIONAL CHANNELS ((Jay D. Pal, Carlos CHANNELS. ((D. Cao, G. Lin, E.M. Westphale, E.C. Beyer, and T.H. Steinberg)) Oberti, Lisa Ebihara.)) Finch University of Health Sciences/The Chicago Department ofMedicine, Washington University, St. Louis, MO 63110. Medical School We have examined the effect of changing external calcium on rat Cx46 and Insulin-mediated increases in cytosolic calcium are synchronized among the cells in chick Cx56 hemi-gap junctional currents expressed in Xenopus oocytes pre- with a Cx38 antisense external calcium from a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells treated oligonucleotide. Reducing 2 mM to 10-7 M caused a remarkable increase in the of the current express the gap junction protein connexin43 (Cx43) and are functionally coupled. magnitude and altered the activation and deactivation kinetics. The amount of Ca++ Thus gap junctional communication is a likely mechanism for the synchronization of required to block the macroscopic current by 50% (IC5o) was 5E-6M and 2E- calcium transients among islet cells. To define the mechanism by which pancreatic 5M for Cx56 and Cx46, respectively. The effect of changing external calcium islet cells coordinate calcium responses, we studied mechanically-induced on the initial I-V relationship of Cx46 and Cx56 hemi-gap junctional currents intercellular calcium waves in the communication-deficient rat insulinoma cell line was also examined. Ca++ blocked the initial current in a voltage-dependent in cells transfected with the RINm5f (RIN), and RIN gap junction protein manner. This effect was more pronounced for Cx56 than for Cx46 and resulted connexin43 (RIN/Cx43). Both RIN and RIN/Cx43 propagated calcium waves that in marked outward rectification of the I-V relation for Cx56 at physiological required release of calcium from intracellular stores, did not involve gap junctional calcium concentrations(1-2 mM). The voltage dependence of block suggests communication, and appeared to be mediated by autocrine activity of secreted ATP that calcium may be binding inside the pore. At the single channel level, acting on P2u purinergic receptors. In addition, RIN/Cx43 cells propagated gap increasing external calcium concentration reduced the probability of opening junction-dependent calcium waves that did not involve release of calcium stores, but of hemi-gap junctional channels, shortened the open time duration in a volt- instead were propagated by influx ofextracellular calcium through voltage-gated age dependent manner and shifted the steady-state activation curve to more calcium channels. The gap junction-dependent intercellular calcium waves were positive potentials. No significant change in single channel conductance was inhibited by preventing plasma membrane depolarization. These studies demonstrate observed. Supported by NIH grant EY10589. two distinct pathways by which insulin-secreting cells might co-ordinate cytosolic calcium rises; and show that it is by ionic traffic and "electrical coupling" that gap junctional communication mediated by Cx43 synchronizes calcium-dependent events in these cells.

533 534 INTERCELLULAR Ca' SIGNALING BETWEEN AIRWAY EPITHELIAL AND USE OF SEQUENCE SPECIFIC ANTIBODIES TO STUDY GAP JUNCTION- SMOOTH MUSCLE CELLS. ((Lisa K. Moore and Michael J. Sanderson)) Dept. of MEDIATEDINTERCELLULARCa2eSIGNALLINGINAIRWAYEPITHELIALCELLS. Physiology, University of Massachusettes Medical Center, Worcester, MA 01655. ((Scott Boitano, *W. Howard Evans, and Ellen R. Dirksen)) Departments of Neurobiology, UCLA School of Medicine, Los Angeles, CA 90095 and *Medical Biochemistry, University of Wales, College of Medicine, Heath Park, Cardiff, U.K. CF4 4XN. In the respiratory tract, smooth muscle cells (SMC's) regulate airway tone while epithelial cells (EC's) support mucociliary clearance to maintain healthy airways. Mechanical stimulation of a single cell in primary cultured airway epithelial cells induced These functions require coordinated multicellular activity which, in EC's, can be a communicated release of Ca2e from IP3-sensitive stores. To investigate the role of gap junctions in this multicellular response, we used pulsed high frequency electroporation and coordinated by the diffusion of IP3 through gap junctions (GJ's) (J.Cell Sci 108; digital imaging microscopy with the Ca2-sensitive dye Fura-2 and evaluated the effect of 2583,1995). We propose 1) that IP3- and GJ-dependent cell-cell signaling is sequence-specific connexin (gap junction) antibodies on intercellular Ca2' waves. important for communicating information about events occuring in the airway to the Electroporation of antibodies to the cytosolic loop (Des 5, generated to AA 108-119, and underlying SMC's and 2) that this signaling provides a mechanism to control airway Des 1, AA 102-112 + 116-124), or to the carboxyl tail (Gap 9, AA 264-283) of connexin caliber. We have established co-cultures, using explant techniques, containing both 32 (Cx32) attenuated or inhibited the mechanically-induced intercellular communication. The effect of Des I EC's and SMC's. The two cell types can be distinguished morphologically and with inhibitory antibody was eliminated by the co-loading of a peptide derived from the cytosolic loop sequence (Des 5 peptide). Conversely, co-loading the Des antibodies for and studies cell-specific keratin, a -actin, myosin. Although previous 5 peptide with the Gap 9 antibody did not alter the inhibition of intercellular signalling. have shown the presence of GJ plaques, the specific GJ proteins expressed in airway Electroporation of antibodies raised to peptide sequences inherent in the extracellular loop SM or EC's have not been fully identified. We have demonstrated with (Gap 11, AA 151-187), or the amino terminus (Gap 10, AA 1-21) of Cx32 did not inhibit immunohistochemical and Western blot techniques that EC's and SMC's express the the mechanically-induced intercellular communication. Also, antibodies raised to peptide of the of Cx43 GJ protein Cx43 and that the amount of Cx43 expression varies both with culture sequences cytosolic loop (Gap 15, AA 131-142) or Cx26 (Des 3, AA 106- 1 19) did not inhibit propagation of intercellular Ca2+ waves in the airway epithelial cells. in SMC's was after 2 weeks in culture age and location. Cx43 expression greatest Western blots using Des I and Gap 10 antibodies recognized proteins with migration on a and in cells at the culture edge. We failed to detect Cx32 in either cell type. We have 12% SDS gel consistent with the migration of Cx32. Conversely, Gap 15 antibodies did demonstrated with digital fluorescence microscopy propagation of mechanically- not display specific binding under similar conditions. This evidence supports the initiated intercellular Ca waves from EC'3 to SMC's and from SMC's to EC's. hypothesis that mechanically-induced Ca2' waves in the airway epithelium are propagated via gap junction formed by Cx32 proteins. It also demonstrates the efficacy of anti-peptide wave is not affected an wxtracellular fluid flow. These data Ca++ propagation by antibodies as specific blockers of gap junction function and for probing specific functional support the hypotheses that 1) Ca++ wave propagation between EC and SMC's regions of the connexins that oligomerise to create the channels facilitating the intercellular occurs via gap junctions consisting of Cx43 ans 2) that the airway epithelium can exchange of physiological signals. S.B. is a Parker B. Francis Fellow. Supported by the Tobacco regulate and coordinate the contraction of SMC'; in response to luminal stimuli. Related Disease Research Program of the University of California, NASA Microgravity Research Program This work supported by HL 49288 to MJS and Parke B Francis fellowship award to LKM. (S.B. and E.R.D.), and the Medical Research Council (W.H.E.).

535 536 PHOSPHORYLATION OF CX43 IN MITOTIC PHASE CELLS. MCDEL FCR GATING OF CCNNEXcLN43 CHANNELS VIA PDSPHDRYIATICZ: POSSIBLE ((P.D. Lampef,W.E. Kurata*, and A.F. Lau*)) Fred Hutchinson Cancer CYTOPLASHIC LOOP-TAIL INrERACTICN. ( (M. M. Shah, J. C. Duncan, A-M Research Center', Seattle, WA 98109; Cancer Research Center of Hawaii*, Martinez, K. A. Balli, and W. H. Fletcher) ) Dapartsents of Physiology and Anatony, J. L. Pettis VAMC, and Lana Linda University School of University of Hawaii, Honolulu, HI 96813 Medicine, Lana Linda, CA 92354. During mitosis the nuclear envelope breaks down, cyclin B dependent We have previously demonstrated that cacmexin43 (Cx43 ) gap kinases become active, cells round up, and intercellular communication levels junction channels can be rapidly and reversibly regulated in vivo by drop. We have sought to investigate whether phosphorylation of Cx43 could microinjection of purified protein kinases. Phosphorylation by pkcA to favor be a of seems channel opening, whereas the effect of pkC depends on possible mechanism regulation of gap junction turnover during the existing phosphorylation state of Cx43 protein. These mitosis. Cx43 immunofluorescence of untreated Normal Rat Kidney (NRK) phosphorzylations are thought to occur on seine residues in the cells shows distinct labeling at cell-cell interfaces and in a perinuclear cytoplasmic carboxyl tip of Cx43. We propose that these negatively arrangement. Overnight treatment of NRK cells with nocodazole, which charged phosphoserines interact electrostatically with positively arrests cells in mitosis, produced a loss of Cx43 immunofluorescence at cell- charged lysine residues in the cytoplasmic loop in a antiparallel cell interfaces and a general staining of the cytoplasm with occasional coil-coil motif and that this interaction is responsible for opening Cx43 channels. To test this hypothesis, site directed mutagenesis was distinctly-stained cytoplasmic structures. Cx43 normally migrates as at least 3 used to construct expression plasmids for Cx43 in which serines 364, bands (nonphosphorylated, P1 and P2). If control and nocodazole-treated 368, and 372 were replaced by alanines or lysine's 102, 105, and 108 cells are metabolically labeled with 35S-methionine or 32p,, lysed, and were replaced with glutaminres. Cells transfected with the seine immunoprecipitated with an antibody for Cx43, a distinct reduction in SDS- mutant Cx43 expression plasmid were deficient in cell-cell PAGE mobility was observed in mitotic cells such that nearly all of the Cx43 casmunication as shown by the absence of dye transfer. Further, injection of pkA, or pse had no effect on cell coupling. In contrast, migrated similarly to the P2. This P2 form of Cx43 in mitotic cells was control transfectants containing expression plasmids for wild type phosphorylated solely on serine. Since cyclin B dependent kinases were Cx43 had detectable channels and transferred dye to neighboring cells likely to be active in mitotic cells, we phosphorylated a GST-Cx43 fusion to a level of 30 %. Cells transfected with the triple lysine protein (residues V236-1382) with p34cdc2/cyclin B kinase. The C-terminal replacement expression vector also showed a reduced level of dye region of Cx43 was an excellent substrate for the cdc2 kinase. Two- transfer in comparison to the control cells, despite the presence of abundant Cx43 gap junctions in the membrane. To further test the dimensional phosphotryptic analysis indicated that there was more than one model, constructs are being made wherein serines 364, 368, and 372 serine phosphorylated in vitro by the cdc2 kinase. We are identifying the sites have been replaced with aspartates or lysines 102, 105, asd 108 are of phosphorylation in Cx43 isolated from mitotic cells and we are studying the replaced with glutanates. These constructs will be used to further significance of these phosphorylation events for Cx43 function. evaluate the effects of protein kinases and phosphatases on the regulation of channel gating. Supported by the Veterans Administration Medical Research, and Lana Linda University School of Medicine. Sunday. Gap Junctions I (537-540) 93a '537 538 TYROSINE AND SERINE PHOSPHORYLATION OF CONNEXIN43 HAVE UBIQUITINATION OF THE GAP JUNCTION PROTEIN CONNEXIN43 INDEPENDENT GATING MECHANISMS. ((A.P. Moreno, B.J. Nicholson)) MAY INVOLVE PROTEIN NEVER Department of Biological Sciences, SUNY at Buffalo. Buffalo NY (Cx43) ASSEMBLED INTO GAP 14260. JUNCTIONS. ((E.L. Hertzberg, T.C. Chan, C. Roy and J.I. Nagy)) Dept. of Gap junctional communication mediated by connexin43 (Cx43), can be modified Neuroscience, Albert Einstein College ofMedicine, Bronx, NY 10461. through phosphorylating agents. Tyrosine phosphorylation, induced by expression of pp6O0*c, uncouples Xenopus oocytes and mammalian cell pairs in minutes. The ubiquitin/proteosome pathway degrades many soluble proteins and, per- Serine/threonine phosphorylation by PKC, has been shown to induce a modulatory haps, a few membrane proteins. Laing and Beyer (JBC (1995) 26399-403) effect, where the unitary conductance distribution of the gap junction channels is presented evidence for of the gap modified to smaller conductances. Here we show that the induction of Tyrosine ubiquitination junction phosphoprotein phosporylation uncouple fast and effectively mammalian cells, without modifying the Cx431 and suted its degradation by the ubiquitin/proteosome pathway. distribution of unitary conductances of Cx43. Topological a~energetic difficulties for degradation of membrane proteins Two types of cells were used in this study: NRK, used as a control, and NRK cells by this pathway are greater for gap junctions where uptake of double transfected with a TS avian sarcomavirus (LA25 cells). Lucifer Yellow transfer assay membrane vesicles annular in cross section has been proposed as a step in the shows that LA25 cells uncouple in A35 min after being placed at room temperature. degradative process. Using an antibody specific for unphosphorylated This time course is not affected after PKC activation Cx43, (30 min in 100nM TPA). The a 47 kDa protein corresponding in size to monoubiquitinated Cx43 total conductance between the cells, as measured with a dual whole cell voltage observed by Laing and Beyer was detected on Western blots of Cl-9 clamp protocol, follows this same trend. Unitary conductance (yj) distribution of NRK cells treated with gap junction channels is similar to the one reported for Cx43 cDNA transfected ALLN, an inhibitor of the ubiquitin/proteosome degradation pathway. Hence SKHepl cells, where a clear dominance of 90-100 pS events is present. at least some ubiquitinated Cx43 is unphosphorylated. Since phosphorylation Yj distribution between LA25 cells at 40 0C is identical to the control NRK's. of Cx43 takes place, at least in part, prior to its assembly into gap junctions After 25 min at room temperature, where Tyrosine phosphorylation has been and Cx43 turnover does not appear to involve dephosphorylation, activated, and a few channels remain open, yj distribution between LA25 cells has ubiquitination of unphosphorylated Cx43 might occur prior to phosphoryla- not been modified, indicating that PKC has not been activated under these tion and assembly. Ubiquitinated Cx43 might even arise from protein circumstances,in the time frame studied. These observations indicate that the gating misfolded as early as translation. Other antibodies specific for Cx43 peptides mechanisms for reduction of gap junction conductance by means of phosphorylation (amino acids 241-260 and 346-360) suggest that the region of amino acids of tyrosine or serine/threonine residues in this system are the former independent, 346-360 contains an closing the gap junction channels but not reducing their unitary conductance. epitope blocked by ubiquitination, perhaps because Iys345 or Iys346 is a site ofubiquitination.

539 540 A NEUROFILAMENT ANTIGEN IS ASSOCIATED WITH LINEAR AND TGF-,31 INDUCES ACCUMULATION OF CONNEXIN43 IN ANNULAR GAP JUNCTIONS IN RAT IRIS AND ENAMEL ORGAN LYSOSOMAL COMPARTMENTS IN ENDOTHELIAL CELLS ((D.M. ((A.R. Hand and D.W. Laird.)) Dept. of Pediatric Dentistry, Univ. of CT Larson, T.G. Christensen, M.J. Wrobleski, G.D.V. Sagar, and E.C. Beyer.)) Health Ctr., Farmington, CT, 06030, and Dept. of Anatomy and Cell Mallory Inst Pathol, Boston Univ Sch Med, Boston, MA 02118 and Dept Biology, McGill Univ., Montreal, Canada, H3A 2B2 Pediat, Washington Univ Sch Med, St. Louis, MO 63110 The function of annular gap junctions is unclear, but experimental evidence We have been studying the relationship between cell growth and expression suggests that annular junctions arise from the internalization of gap junction of the gap junction protein Connexin43 (Cx43) in cultured bovine aortic en- membranes. Thus, annular junctions may represent one of the first stages of dothelial cells. As part of these studies, we have examined the effect of the gap junction turnover and degradation. However, the mechanisms involved growth inhibitory cytokine TGF-B31 on Cx43 expression. TGF-,B treatment in formation and internalization of these junctions are unknown. The ep- caused an increase in Cx43 mRNA and synthesis, content, and half-life of the ithelium of the iris and cells of the papillary layer of the rat enamel organ protein within 24h. Immunostaining for Cx43 showed the characteristic punc- (EO) have numerous linear and annular gap junctions. Some of the EO an- tate and linear expression along cell-cell contacts, together with Golgi stain- nular junctions have concentric layers of junctional membrane with irregular ing, both of which were more intense in the subconfluent treated cells than in outlines surrounding condensed cytoplasm, suggesting that they may be un- controls. After 2d of treatment, vesicular staining developed in some of the dergoing degradation. Immunogold labeling of Lowicryl sections of both iris treated cells; these vesicles increased in frequency and size with time. At 6d, a and EO, and cryosections of iris, showed strong reactivity of the junctions variable proportion of cells contained large, intensely staining vesicles, filling with an anti-connexin43 antibody. The cytoplasmic faces of linear and espe- much of the perinuclear cytoplasmic space. This high concentration of vesic- cially annular junctions also showed reactivity with an antibody to a 160 kD ular Cx43 may explain the increased content and half-life we have observed neurofilament polypeptide. The junctions were unreactive with anti-keratin in the treated cells. The vesicles may be lysosomes since a similar pattern antibodies, although adjacent intermediate filament bundles in EO cells were was found using a fluorescent probe for acidic compartments. TEM revealed labeled. Western blots confirmed reactivity of the anti-neurofilament antibody prominent lysosomal figures with considerable heterogeneous material. The with a 160 kD protein in a rat brain extract; no cross-reactivity was seen with appearance of intact (based on immunoreactivity in situ and in immunoblots) connexin43. These results indicate that cytoskeletal components are associ- vesicular Cx43 suggests decreased degradation. It appears that TGF-l3 not ated with gap junctions in some tissues. These components may function only upregulates Cx43 expression in endothelial cells but may interfere with to stabilize junctional structure and/or participate in the internalization of its degradation. (Supported by USPHS grants HL52697 to DML and HL45466 annular junctions. Supported by UCHC and MRC of Canada. to ECB)

Structure of the Nuclear Envelope (541-542)

541 542 DYNAMICS OF NUCLEAR STRUCTURES AND THEIR NUCLEAR CHARACTERIZATION OF THE PROCESSES AND VESICLE TYPES TRANSPORT ACTIVITY VISUALIZED IN LIVING MAMMALIAN INVOLVED IN THE FORMATION OF A NUCLEAR ENVELOPE ((D. CELLS BY FLUORESCENCE MICROSCOPY. ((Tokuko Haraguchi, Lourim and G. Krohne)) Department ofElectron Microscopy, Theodor Boven Toru Kanedal,2, Takako Koujinl and Yasushi Hiraokal)) 1 Kansai Advanced Institute, University ofWurzburg, D-97074 Wfirzburg, Germany. Research Center, Communications Research Laboratory, Kobe, Japan, 2 Olympus Optical, Co., Ltd., Tokyo, Japan. UsingXenopus egg extract we have investigated the formation ofthe nuclear envelope and associated structures at the biochemical and ultrastructural levels Dynamics of breakdown and remodelling of nuclear structures during mitotic using EM and a variety of immunological tools. Using isoform-specific lamin cycles were examined in living HeLa cells by the use of a computerized antibodies we have identified and quantified three B-type lamins present in the fluorescence microscope system with a precise temperature control. Fluorescein-conjugated nucleoplasmin (Fl-NP) and rhodamine-conjugated Xenopus oocyte and egg. Relative to lamin L1, lamins L1 and L5 are present in Xenopus oocytes at ratios of 1:100 and 1:10, respectively. However, tubulin was microinjected into cells that had been stained with Hoechst 33342. the synthesis Triple-wavelength or double-wavelength time-lapse analysis of fluorescently of lamin LI protein is elevated during egg maturation, resulting in roughly a 10-fold stained cells was carried out at 37 'C. Microinjected Fl-NP was transported increase. Only five to ten percent of each lamin isoform is associated with an into an interphase nucleus within 10 min. and acted as a marker of a nucleus, isoform-specific population of egg vesicles, the vast majority ofwhich appear At prophase when nuclear envelope was still present, Fl-NP started leaking separate from vesicles containing gp200, an integral pore protein. Our data from nucleus to cytoplasm, suggesting that partial breakdown or functional suggest that at least four independent populations of Xenopus egg vesicles are change of nuclear envelope takes place at this stage. Double immuno- involved in the formation of a nuclear envelope. Surprisingly, lamin polymerization fluorescence staining of nuclear lamin and tubulin in fixed specimens revealed in fractionated extract does not depend on the presence ofmembranes or that at this stage nuclear was envelope invaginated and mitotic spindle was chromatin. In fact, when an ATP-depletion system or membrane fusion inhibitors observed along the nuclear invagination. At prometaphase, Fl-NP was spread such as nonhydrolyzable GTP analogs are added to complete egg extracts, lamin out into cytoplasm upon the nuclear envelope breakdown, and then relocated appears from cytoplasm to chromosomes at early telophase when chromosomes were polymerization to be enhanced. Moreover, in the presence of exogenous still condensed. In fixed specimens corresponding to this stage, nuclear ATE or GTE, we have observed a decrease in both lamin polymerization and the lamina network was not formed yet except for region near microtubule- formation of annulate lamellae, the pore containing structures formed in egg attachment sites. Live analysis of Fl-NP is a powerful tool to study nuclear extracts when suboptimal amounts ofchromatin substrates are present. Our data transport activity of the nucleus. We will present these results on a video indicate that lamin polymerization, vesicle fusion and pore formation are regulated movie. (Please refer to another abstract of Haraguchi et al. in this meeting for by different mechanisms. We can now begin our analysis oflamin polymerization tubulin and chromosome dynamics.) and the mechanism(s) which target vesicles to the surface of chromatin. 94a Structure of the Nuclear Envelope (543-548). Sunday 543 544 Identification of novel Xenopus nucleoporins using in vitro assembled STRUCTURAL INTERMEDIATES IN NUCLEAR PORE COMPLEX annulate lamellae. ((B. R. Miller, M. A. Powers, and D. J. Forbes)). ASSEMBLY IDENTIFIED BY SCANNING EM. Department of Biology, University of Califomia at San Diego, La Jolla, CA ((C. Wiese, M.W. Goldberg*, T.D. Allen*, and K.L. Wilson)) Cell 92093. Biology & Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD, USA. *CRC Dept. Structural Cell Biology, Paterson Annulate lamellae (AL) are stacks of flattened membrane clsternae Institute for Cancer Research, Christie Hospital National Health Service perforated by numerous nuclear pore complexes (NPCs). We have used UK. AL assembled in vitro from Xenopus egg extracts as a simplified system in Trust, Manchester, which to study nuclear pore composition and assembly. We have now developed protocols for obtaining highly purified NPCs from the AL Nuclei were assembled in vitro using fractionated extracts ofXenopus assembly reactions. In the most ennched fractions, knowopore proteins are eggs, and examined at different times during assembly by Field Emission readily Identifiable on silver stained gels. Since the purificAW protocol does In-lens Scanning Electron Microscopy (FEISEM). FEISEM can resolve not require strong chaotropic agents, we believe that these purified pores biological samples to within 2-5 nm. The flattening of larger nuclear should contain a complete complement of nucleoporins, and should allow for vesicles against the chromatin surface required a cytosolic factor, the generation of a representational library of monoclonal antibodies against provisionally named 'flattening factor'. We hypothesize that membrane pore proteins. flattening may be a prerequisite for the intra-lumenal fusion events that We have also utilized the assembly of AL as a simplified and rapid biochemical generate the pores in which nuclear pore complexes (NPCs) assemble. assay for the identification of novel nucleoporins. Lectin blots We found that NPCs can assemble in about 8 minutes in vitro. FEISEM using labeled wheat germ agglutinin (WGA) strongly detect only the known of nuclear glycosylated Xenopus nucleoporins, nup6O, nup97 and nup200. images newly-assembled, growing envelopes revealed However, structures scattered mature there are considerably more proteins from the egg extract which are interesting among the NPCs: rare 'dimples' specifically retained on immobilized WGA. By biotinylation of this mixture of (outer membrane depressions, 5-35 nm diameter), more abundant holes proteins, we have introduced an easily detectable label with which to monitor (pores) with various edge geometries (35-45 nm diameter-, 3.3% of their distribution. When biotinylated WGA-binding proteins are added to AL structures), pores with 1-8 triangular 'star-ring' subunits (2.1% of assembly reactions, we can observe at least two previously unidentified, structures), and more complicated pore-associated structures. Empty biotinylated proteins which are greatly enriched in the AL pellet. Further pores, star-rings, and other intermediates accumulated preferentially when fractionation experiments indicate that these novel proteins co-purify with nuclei were treated with Wheat Germ Agglutinin (WGA) or BAPTA, nuclear pores and are therefore strong candidates for new Xenopus suggesting that these structures are intermediates in NPC nucleoporins. We are presently in the process of further characterization of assembly. these new putative nucleoporins.

545 546 DOMAIN SPECIFIC DISASSEMBLY AND REASSEMBLY OF NUCLEAR SEQUENTIAL ASSEMBLY OF LBR AND LAMIN B DURING SEA URCHIN MEMBRANES IN MITOTIC CELLS. ((B. Buendia and J-C. Courvalin)) Departement MALE PRONUCLEAR ENVELOPE FORMATION IN VITRO. ((P. Collas', J.-C. de Biologie Cellulaire, Institute Jacques Monod, CNRS, Universitd Paris 7, Paris, and D.L. France. Courvalin2, Poccia3)) 'Dep. of Biochemistry, Norwegian Coll. Vet. Med., Oslo, Norway; 2Inst. J. Monod, CNRS, Univ. Paris VII, Paris, France; 3Dep. of The nuclear envelope contains three distinct membrane domains, the outer Biology, Amherst College, Amherst, MA 01002. membrane, the inner membrane and the pore membrane, that reversibly vesiculate in mitosis. In a previous study, we suggested from single immunofluorescence analysis We have identified a protein p56 in sea urchin gametes which cross-reacts with an of mitotic cells in culture that mitotic vesiculation of the nuclear membranes may antibody raised against the N-terminal domain of human lamin B receptor (LBR). proceed in a domain-specific manner. In the present study, we add biochemical LBR is a protein of the inner nuclear membrane with chromatin and lamin B support to this hypothesis by sorting domain-specific mitotic vesicles. Antibodies binding activities. is at the and sea directed against LBR, a marker of the inner membrane, and glycoprotein gp2l0, a p56 present exclusively tip base of the urchin sperm marker of the pore membrane, were used to isolate by affinity two populations of nucleus from which it is rapidly removed by egg cytoplasmic extracts. During mitotic vesicles that were selectively enriched in each of these markers. These two subsequent formation of the male pronuclear envelope in vitro, membrane vesicles vesicle populations were of different size distribution, the pore derived vesicles being (MVs) containing p56 bind to the chromatin surface and fuse, distributing p56 less variable in size and smaller (< 200 nm) than the inner membrane derived vesicles. around the nuclear periphery. p56 is located in different MVs from lamin B, which Double immunofluorescence analysis of mitotic cells in culture showed that the time also exists in a soluble pool. Both p56 and lamin B vesicles are found in a fraction course and topology of disassembly and reassembly of inner and pore membrane of membranes domains were different confirming that domain-specific vesicles are generated during cytoplasmic (MV2B) which contributes the bulk of the nuclear mitosis. In these studies, protein LAP2/thymopoietin A, another marker of the inner envelope (NE). p56 but not lamin B is required for binding of MV2B vesicles to nuclear membrane, was segregating as LBR suggesting that both proteins were chromatin. Only after formation of the NE is lamin B incorporated into the nuclear contained in the same mitotic vesicles. From this study, we conclude that the domain periphery, largely from the soluble pool. Incorporation of lamins from the soluble differentiation of the nuclear membrane is maintained in mitotic vesicles. pool and additional MVs are required for further swelling of the nucleus. Only after incorporation into the NE do p56 and lamin B physically associate. We propose that p56 is a sea urchin lamin B receptor because it is a 56 kDa integral membrane protein which reacts with anti-LBR and binds both to chromatin and to lamin B. Its incorporation into the NE prior to lamin assembly suggests a primary role in formation of a complete NE. p56 may tether the inner membrane to chromatin and subsequently to the lamina.

547 548 NUCLEOPORINS AT FERTILIZATION: PRONUCLEAR DEVELOPMENT STRUCTURE-FUNCTION ANALYSIS OF THE BINDING OF CHROMOSOMAL HP1 REQUIRES THE RECRUITMENT OF NUCLEAR PORE COMPLEXES TO PROTEINS TO THE NUCLEOPLASMIC DOMAIN OF INNER NUCLEAR MEMBRANE PRONUCLEAR ENVELOPE AND THE ASSEMBLY OF ANNULATE LAMELLAE PROTEIN LBR. ((0. Ye, I. Callebaut, J.-C. Courvalin, and H.J. Worman)) Departments (AL) FROM THE PRE-AL, A NOVEL PARACRYSTALINE of Medicine and Anatomy and Cell Biology, Columbia University College of Physicians STRUCTURE. ((P. and New NY Sutovsky, C. R. Simerly. and G. Schatlen)) Department of Zoology, Surgeons, York, and CNRS, Universitd Paris 6 and 7, Paris, France. University of Madison WI Wisconsin, 53706 We have previously demonstrated that the nucleoplasmic domain of inner nuclear membane protein LBR binds to two human proteins, HP1 Hsa and HP1 Hay, which Mammalian sperm lose their are membranes and nuclear envelope homologous to the Drosophila heterochromatin protein HP1. We derived structural (NE) at fertilization and male pronucleus (PN) development is delineated information from 2D representations of LBR and human HP1 proteins using by a de novo formation of pronuclear NE. We demonstrate that the hydrophobic cluster analysis. This analysis demonstrated that the nucleoplasmic fertilizing sperm induce the assembly of cytoplasmic annulate lamellae domain of LBR is comprised of three stuctural subdomains: a globular subdomain (AL) and the recruitment of nuclear pore complexes (NPC) to the from amino acids 1 to 60, an extended loop subdomain from between amino acids 60 and 150 and a second pronuclear NE shortly after sperm incorporation. Migration of NPC to globular subdomain from amino acids 150 to 208. Analysis of the two human HP1 proteins showed them to also contain a tripartite with NE, but not the assembly of AL, is sensitive to Ca2+ chelation, which structure a globular region containing the chromodomain at the amino-terminus, a blocks male PN formation as does charged the intracytoplasmic injection of the cluster in the center, and a second globular region with structural similarity to the first NPC-antagonist wheat germ agglutinin (WGA). The dynamics of NPC in at the carboxyl-terminus. Each of the LBR subdomains, and larger and smaller fertilized eggs can in part be mimicked by the parthenogenetic activation portions, were tested in the yeast two-hybrid assay for interaction with HP1 proteins. with ionomycin or ionomycin/6-DMAP. Sperm-oolemma binding, but not The portion of LBR from amino acids 97 to 208 was the smallest stretch able to its entry into egg cytoplasm is necessary to induce AL interact with the HP1 proteins. This region of LBR contains a threonine residue that is and NPC assembly, in as shown by the treatment with B. specifically phosphorylated mitosis when the nuclear membranes dissociate from cytochalasin The assembly of AL occurs the chromosomes. In the yeast two-hybrid assay, in the carboxyl-terminal globular a paracrystaline precursor structure associated with the Golgi, the subdomains of the human HP1 proteins interacted with the nucleoplasmic domain of pre-AL. Targeting of AL towards the pronuclei and their subsequent fusion LBR and with the stretch of LBR from amino acid 97 to 208. These results show that with NE suggest that AL actually are the precursor of NPC. The assembly the carboxyl-terminal globular regions of HP1 proteins interact with the carboxyl- of AL and NPC therefore represent a specific, previously unrecognized terminal portion of the nucleoplasmic domain of inner nuclear membrane protein LBR. step in the activation of the mammalian oocyte at fertilization, that appears to be required for pronuclear development. Supported by grants from NIH and USDA. Sunday. Structure of the Nuclear Envelope (549-554) 95a

549 550 DIFFUSIONAL MOBILITY OF AN INNER NUCLEAR MEMBRANE BIOCHEMICAL AND MOLECULAR ANALYSIS OF THE MAMMALIAN PROTEIN IN MAMMALIAN CELLS. ((Jan Ellenberg, John F. Presley, NUCLEAR PORE COMPLEX AND THE REGULATION OF NUCLEAR Kristien J.M. Zaal, Howard J. Worman* and Jennifer Lippincott-Schwartz)) TRANSPORT ((Michael J. Matunis and Gunter Blobel.)) The Rockefeller NICHD, CBMB, National Institutes of Health, Bethesda, MD 20892. * College of Physicians & Surgeons of Columbia University, New York, NY 10032. University The mammalian nuclear pore complex (NPC) is 125 MDa supramolecular assembly The nuclear envelope consists of two concentric nuclear membranes that that forms the portal for entry and exit of macromolecules to and from the nucleus. merge at insertion sites of nuclear pore complexes (NPC). The outer nuclear Structurally, the NPC consists of two symmetrical halves (with respect to the plane membrane is continuous with the endoplasmic reticulum (ER) and as a conse- of the nuclear envelope), each half displaying eight-fold rotational symmetry and to- quence the nuclear lumen is continuous with the ER lumen. In spite of this in- gether forming a cylinder with a central pore of approximately 40 nm in diameter. The timate relationship, nuclear envelope and ER are functionally and structurally entire NPC is thought to be comprised of up to 100 unique proteins (nucleoporins). distinct and inner and outer nuclear membranes (INM/ONM) contain different We have undertaken a systematic fractionation of the rat liver nuclear envelope with sets of proteins. The mechanism of sorting and retention of membrane proteins the goal of obtaining precise information relating to the molecular composition and to the [NM remains obscure. Models for this process include free or limited organization of the NPC. Using a series of novel extraction conditions, we have defined diffusion between ONM and INM through aqueous channels along the side of the protein composition of the marrunalian NPC for the fist time. Intermediates in the ATP driven NPC, active transport along the edge of the NPC, vesicle medi- the fractionation procedure, including nuclear envelopes, highly enriched nuclear en- ated transport between ONM and INM, as well as sorting upon reassembly of the nuclear envelope after mitosis. Retention of resident INM proteins might be velopes, pore complex lamina fractions, and extracted pore complex lamina fractions, conferred by binding to nuclear ligands (lamins, chromatin...) and/or formation were monitored by electron microscopy and by immunoblot analysis, demonstrating of homo/hetero-oligomers between INM proteins. To investigate these possi- the integrity of the pore complex up to the final step in the fractionation. In the bilities we have tagged a resident protein of the INM, the lamin B receptor, with final step, NPCs were extracted from the pore complex lamina fraction and found to the green fluorescent protein (GFP). By confocal microscopy in living cells we contain all of the presently known nucleoporins, and no more than 50 unique pro- found a localization of the chimeric protein (LBR-GFP) to the nuclear envelope teins in total. Of these 50 unique proteins, 18 could be identified with characterized and the ER, consistent with the distribution of the native protein (Soullam and nucleoporins, or NPC-associated nuclear transport factors. We are currently pur- Worman, 1995). LBR-GFP was concentrated to evenly spaced domains in the suing several approaches to further characterize the purified nucleoporins. Ligand INM, reminiscent of funcional clusters reported for several nuclear proteins. blot analysis is being used to identify interactions between nucleoporins and soluble Examination of the diffusional mobility of LBR-GFP by photobleaching tech- nuclear transport factors. Blots of purified nucleoporins probed with radio-labeled niques, including FLIP and FRAP (Cole et al, 1996), indicated high mobility of Ran-GTP has identified several Ran-interacting proteins, including Nup358 and a LBR-GFP in the ER and significantly reduced mobility of LBR-GFP in INM novel fugl (RanGAPI)-like protein. In addition, interactions between the FXFG clusters. Moreover, repetitive photobleaching of only the ER did not deplete repeat-containing nucleoporins and the Ran-GDP binding transport factor, pl, have the pool of LBR-GFP in the INM, suggesting restricted movement of LBR be- also been discovered. This information is being integrated to develope testable models tween INM and ONM/ER. in the diffusional of Changes mobility LBR-GFP that describe the translocation of molecules pore. within INM and ONM/ER during the cell cycle are currently being investigated. through the nuclear

551 552 A NOVEL PROTEIN WITH FG REPEATS LOCALIZED TO THE IDENTIFICATION OF A NUCLEAR PORE LOCALIZATION DOMAIN FOR THE NUCLEOPLASMIC SURFACE OF THE NUCLEAR PORE NUCLEOPORIN TPR: POSSIBLE PHOSPHORYLATION-DEPENDENT COMPLEX. ((T. Guan, F. Fan*, L. Gerace, N. Arnheim*)) ASSOCIATION WITH THE NUCLEAR PORE COMPLEX. ((P.L. Bangs, E.G. Fey, and S.J. Doxsey)) Department of Cell Biology and Program in Department of Cell and Molecular Biology, Scripps Research Molecular Medicine, University of Massachusetts Graduate School of Institute, La Jolla, CA 92037. Department of Biological Sciences, Biomedical Sciences, Worcester, Massachusetts 01605. University of Southern California, Los Angels, CA 90089 The nucleoporin TPR (nup265) is a component of the cytoplasmic A rat testis fibrils that extend from the nuclear pore complex (NPC). Naturally cDNA has been cloned that encodes a 40 kDa protein occurring chimeric proteins that consist of the N-terminal 200 amino with 5 FG (phe, gly) dipeptide repeats. Immunofluorescence acids of TPR fused to the protein kinase domains of the MET, TRK and microscopy had indicated that this protein (Rtp6O), which migrates RAF oncoproteins transform cell lines suggesting that the TPR at about 60 kDa on SDS gels, is localized at the nuclear envelope sequences relocate the kinases to the NPC. However, HA-tagged in a nuclear pore complex-like distribution in mammalian polypeptides comprised of the first 383 amino acids of TPR, when spermatocytes and cultured cells. Using affinity purified antibodies overexpressed in several cell types, are cytoplasmic and detergent to Rtp6O, we have detected a homologous antigen in isolated rat extractable These results indicate that the oncogenic activity of TPR- liver nuclear electron protein kinase chimeras is not due to a mislocalization of a protein envelopes by immunoblotting. Immunogold kinase to the NPC. In fact, the NPC localization domain of TPR is microscopy of liver nuclear envelopes shows that this protein is contained within amino acids 383-734. Interestingly, this domain is localized specifically on the nucleoplasmic surface of nuclear pore phosphorylated in vivo possibly in an NPC localization-dependent complexes. Gold labeling is found at an average distance of 35-40 fashion. All HA-tagged chimeras that contain this stretch of amino nm from central plane of the NPC. This indicates that the Rtp6O acids localize to the NPC and also form ring-shaped cytoplasmic foci. antigen is more peripherally located than another FG-repeat Endogenous TPR localizes to these foci, and other components of the nuclear pore protein (the p62 complex), but is more centrally NPC may do so as well. Immunoprecipitation of the expressed 1-383 situated than the FG-repeat containing Nupl 53. Rtp6O has a polypeptide of TPR indicates that it forms strongly associated homo- of residues to other oligomers and perhaps heteropolymers with other components of the unusually high percentage charged compared nuclear transport apparatus. We are currently investigating these FG repeat nuclear pore complex proteins, and is expected to have structures and the mechanism and regulation of TPR's association distinctive properties. The functional analysis of this component is with the NPC. underway.

553 554

NUCLEAR BINDING OF HERPES SIMPLEX VIRUS TYPE 1 CAPSID NUCLEAR PORE COMPLEX PROTEINS IN PLANTS ((A. Heese-Peck and ((Paivi M. Ojala, Beate Sodeik, Melanie Ebersold, and Ari Helenius.)) N.V. Raikhel)) DOE-Plant Research Laboratory, Michigan State Department of Cell Biology, Yale University School of Medicine, New Haven, University, East Lansing, Ml 48824-1312. CT 06520-8002. After fusion of the herpes simplex virus type 1 (HSV-1) envelope with the The nuclear pore complex (NPCI is the site of nucleocytoplasmic plasma membrane, the incoming capsids are efficiently and rapidly transported transport of macromolecules across the nuclear envelope. In plants, across the cytosol to the nuclear pore complexes (NPC). The cytosolic trans- little is known about NPC proteins and their function in nuclear port of capsids in Vero cells is mediated by microtubules. At the NPC, vi- transport. Previously, we showed by electron microscopy that proteins ral DNA is injected into the nucleus where viral replication and assembly at the NPC of tobacco suspension-cultured cells are modified by N- of progeny capsids take place. We have analysed HSV-1 capsid entry using acetylglucosamine (GIcNAc). At least eight of these proteins are biochemical in vivo and in vitro as well immunofluorescence and im- assays as modified terminal GIcNAc as determined munoelectron Anti-NPC antibodies inhibit the accumulation of by by galactosyltransferase microscopy. the with terminal GIcNAc differ from HSV-1 at the NPC in vivo. a assay in assays. Interestingly, plant glycans capsids By using quantitative uncoating the of vertebrate NPC in that they infected cells, we have found that about 40% of the entered incoming capsids single O-linked GlcNAc proteins are have released their DNA at 4 hr post infection. To study the binding of capsids larger in size than five GIcNAc residues. Most of these plant glycans are and the release of the DNA into the nucleus (uncoating) we developed an assay bound to the proteins via an 0-linkage. In these studies, several plant for capsid binding to the NPC in vitro. Purified capsids bound to nuclei of proteins with terminal GIcNAc were purified by lectin affinity permeabilized Vero cells or purified from rat liver in the presence of exogenous chromatography. The most abundant glycoprotein (gp4O) with an cytosol. Electron microscopy demonstrated that the capsids were localized to apparent mass of 40 kD was subjected to tryptic digestion to obtain the NPC, and more specifically, to the cytoplasmic fibers emanating from the amino acid sequence information. A specific product of 600 bp was NPC in this in vitro assay. WGA and anti-NPC antibodies interfered with the produced by polymerase chain reaction (PCR)-based amplification using binding of capsids to the NPC. In addition to the capsid proteins, our purified degenerate oligonucleotide primers. Sequence comparison indicatedthat capsids also contain at least two tegument proteins, namely VP1/3 and VP16. the PCR-fragment is up to 32 % identical to aldose-1 -epimerases. These We are currently analysing the requirements for viral uncoating in the assay. enzymes catalyze the interconversion of a and anomers of certain sugars and have been localized to the nucleus in rat. We are currently producing antibodies against gp4O to investigate the subcellular and subnuclear localization of gp4O in plants. 96a Structure of the Nuclear Envelope (555-556). Sunday 555 556 A 78KDA PROTEIN IS RESPONSIBLE FOR THE TARGETING OF NUCLEAR SORTING OF A MAMMALIAN NUCLEOPORIN TO ENVELOPE PRECURSOR VESICLES TO CHROMATIN UPON EXIT FROM THE YEAST NUCLEAR PORE COMPLEX. MITOSIS ((Sheona P. Drummond, Carl G.W. Smythe and Christopher J. Hutchison.)) Dept. of Biological Sciences, University of Dundee Dept. of ((W. Barth, S. Chatterjee, L. Le, M. Javier and U. Stochaj)) Biochemistry, University of Dundee Department of Physiology, McGill University, 3655 In order to examine the ability of populations of nuclear envelope precursor vesicles to Drummond Street, Montreal H3G 1Y6, Canada bind to chromatin, Xenopusegg extracts, known to contain vesicles with such ability, were fractionated by differential and density gradient centrifugation (see Vigers and Nucleocytoplasmic traffic of macromolecules is mediated Lohka, 1991). This procedure yeilded two vesicle containing fractions, one of which was greatly enriched in vesicles with the ability to bind to chromatin. Biochem- by nuclear pore complexes (NPCs). Mechanisms for traffic ical characterisation of these two fractions indicated that the Xenopus homologue of proteins and RNA between cytoplasm and nucleus are of human ribophorin A was present in both preparations whereas a 78kDa integral conserved from yeast to man. Proteins of the NPC, termed membrane protein (NEPB78) was present only in the fraction containing vesicles nucleoporins, play an essential role for these transport capable of binding to chromatin. Indirect immunofluorescence assays indicated the reactions. To date, pathways that mediate and presence of both proteins in vesicles that were bound to chromatin. The intensity targeting of anti-NEPB78 immunofluorescence was maximal at the stage of vesicle binding to incorporation of nucleoporins into the NPC have not been chromatin. In contrast, anti-ribophorin immunofluorescence was maximal only after defined. It is currently not known whether these sorting membrane fusion and expansion. This indicates that while NEPB78 is only incorpo- reactions are conserved among eukaryotes. To address rated at the initial stages of nuclear envelope formation, the ribophorin homologue these questions, we are studying the targeting of is also incorporated during nuclear envelope fusion and expansion. The relative effi- in the S. cerevisiae. ciences of incorporation of these two proteins into the nuclear envelope were analysed nucleoporins yeast We have expressed by SDS-PAGE and quantitative Western blotting. This investigation showed that a mammalian nucleoporin in yeast cells, and we the binding of vesicles to chromatin depletes the incubation miix of NEPB78. In demonstrate that this protein is associated with the yeast addition, when compared with incorporation of the ribophorin homologue, NEPB78 NPC. To determine the minimal region for targeting to the was incorporated into the nuclear envelope with approximately eight times greater efficiency. This further suggests that NEPB78 is specifically associated with vesicles NPC deletion mutants were generated, and the cellular that bind to chromatin durong the initial stages of nuclear envelope assembly. Neu- localization of these truncated proteins was analyzed. We tralisation experiments have shown that pre-incubation of vesicles known to bind to are currently testing whether the synthesis of mammalian chromatin with mAb specific for NEPB78 inhibits their binding to chromatin. This nucleoporins in S. cerevisiae affects nucleocytoplasmic implicates NEPB78 as the nuclear membrane protein required for the targeting of transport of macromolecules across the yeast nuclear nuclear envelope precursor vesicles to chromatin upon exit from mitosis. pore.

Proteins of the Nucleus (557-560).

557 558 1 04KD-DIACYLGLYCEROL KINASE (TYPE IV) CONTAINING ANKYRIN-LIKE LAMIN B IS PHOSPHORYLATED PRIOR TO REMOVAL FROM SEA REPEATS LOCALIZES IN THE CELL NUCLEUS. ((K.Goto and H.Kondo)) URCHIN SPERM NUCLEI IN FERTILIZED EGG CYTOSOLIC EXTRACTS. Department of Anatomy Tohoku University School of Medicine, Sendai ((P. Collas', and D.L. Poccia2)) 'Dep. of Biochemistry, Norwegian College of 980, Japan. (Spon. by J.A. Glomset.) Veterinary Medicine, 0033 Oslo, Norway; 2Dep. of Biology, Amherst College, Amherst, MA 01002. In the process of signal transduction involving phosphoinositide turnover, diacylglycerol kinase (DGK) converts a second messenger DG to The sea urchin sperm chromatin periphery is associated with lamins, including phosphatidic acid (PA). As DG acts an activator of several forms of a 65-kD lamin B. In extracts from fertilized sea urchin eggs, sperm lamins are protein kinase C (PKC), DGK is regarded as an attenuator of the activity removed from nuclei prior to chromatin decondensation. We examined the of PKC. In addition, recent studies have shown that PA might also be a phosphorylation state of the 65-kD sperm lamin B during incubation of nuclei in second messenger that is involved in the of DNA the egg cytosolic extracts depleted of endogenous B-type lamins. Solubilized sperm regulation synthesis, lamin induction of c-myc and c-fos, and cAMP formation. Therefore, DGK is B was recovered by immunoprecipitation from cytosol using the anti-sea urchin lamin B antibody W3-1, and lamin B recovered involved in only in the attenuation of DG, but also in the of a sperm-associated by production extraction of nuclei with 1% TX-100. On blots of nuclei incubated in possible second PA. We have so far cloned three DGK sperm messenger, cytosol for I mn, all sperm lamin B was detected as a 68- kD band. This band was from a rat brain cDNA and characterized their isozymes library enzymatic converted to 65-kD by alkaline phosphatase treatment of nuclei, that the properties and localization. In this we isolated a novel cDNA for DGK indicating study shift was due to sperm lamin B phosphorylation. Between I and 5 minin egg (DGK-IV) from a rat brain cDNA library. This cDNA encoded a protein of cytosol, phosphorylated lamin B was gradually solubilized, and completely 929 amino acids with a calculated molecular weight of 1 04kDa. The removed from nuclei by 8 min. At 10 min, all sperm lamin B wasrecovered from primary structure of DGK-IV was distinct from those of previous DGK-I, -Il the cytosol in an alkaline phosphatase-sensitive 68-kD form indicating that sperm and -ll in that DGK-IV contained no EF-hand motifs but four ankyrin-like lamin B remains phosphorylated for at least 10 minafter removal from nuclei. repeats at the carboxyl terminus. This structural feature of DGK-IV dosely Lamin B phosphorylation and its removal required cytosol and an ATP-generating resembles the recently cloned eye-specific DG kinase of Drosophila which system. Phosphorylation was partially inhibited with the kinase inhibitor DMAP is encoded by retinal degeneration A (rdgA) gene. However, DGK-IV was (1mM). Under these conditions, non-phosphorylated and phosphorylated forms of detected dominantly in the thymus and brain with relatively low lamin B were exclusively recovered fomnuclei and cytosol, respectively. 5 mM expression in the eye. Furthermore, the primary structure of DGK-IV DMAP fully inhibited lamin phosphorylation, its removal from sperm nuclei, and contained a nuclear targeting motif, and DGK-IV was localized in the chromatin decondensation. The results strongly suggest that phosphorylation of nucleus by immunocytochemistry of COS-7 cells transfected with the sea urchin sperm lamin B by a kinase fromegg cytosol is required prior to removal epitope-tagged cDNA, suggesting an involvment of DGK-IV in intranuclear from sperm nuclei. These events precede and may be required for chromatin processes. decondensation in vitro.

559 560 FURTHER CHARACTERIZATION OF A UNIQUE NUCLEAR PORE ISOLATION AND CHARACTERIZATION OF NUCLEOLAR PRE-rRNA- PROTEIN ((D. L. Adams and L.D. Hodge)) Department Cellular CONTAINING RIBONUCLEOPROTEIN COMPLEXES FROM HUMAN CELLS. Biology and Anatomy, Medical College of Georgia, Augusta, GA. ((L. E. Pomeranz and S. Pifiol-Roma)) Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, NY 10029. Previously, we have presented evidence that a new nuclear pore We have undertaken a thorough analysis of the proteins that associate with protein in HeLa Ss cells has been detected by a monoclonal nuclear pre-rRNA and with nuclear products of pre-rRNA processing in antibody (mAb501) which we have raised (Mol. Biol. of the Cell §, vertebrate cells. Our goal is to gain a better understanding of the process of 424a, 1995). It was reasoned that such a protein (p51) was new ribosome assembly, as well as structure-function relationships and dynamics in because of its apparent size (51 kDa), its positioning at or around the the eukaryotic nucleolus. To this end, we have isolated pre-rRNA-containing base of a nuclear and because it remains associated with ribonucleoprotein complexes from human (HeLa) cells, using a rapid pore, immunopurification with chromosomes mitosis for procedure monoclonal antibodies to nucleolin, one of during (submitted publication). Continued the major pre-rRNA-binding proteins. Two-dimensional gel electrophoresis analyses of p51 using mAb501 have included ultrastructural and analysis reveals a discrete set of proteins, in addition to nucleolin, ranging from fluorescent immunocytochemistry, as well as immunoabsorption <14kDa to > 200kDa and a wide range of isoelectric points (<5 to >9). We are from nuclear sonicates prepared in the absence of chaotropic in the process of determining which of these proteins correspond to previously agents. In particular, circular clusters of immunogold are observed identified nucleolar proteins. Some of these proteins have electrophoretic in nuclear and clusters mobility properties characteristic of ribosomal proteins. In addition, some of tangential sections, cytoplasmic resembling the proteins in these react polyribosome with some in the ER are complexes strongly with antisera from patients with synthesis along particles autoimmune diseases. Preliminary analyses reveal the presence of pre-rRNA observed. The latter could explain the previously reported weak as well as processed rRNA in the immunopurified complexes. We have cytoplasmic fluorescence. Isolated metaphase plates and post- obtained antisera by immunization of mice with these RNP complexes metaphase separated sets of chromatids (prenuclei) retain p51 prepared by large-scale immunopurification in order to generate monoclonal which is relocated after chromatid coalescence. Immunoabsorbed antibodies to novel pre-rRNP proteins. Immunofluorescence microscopy on p51 was WGA and co-isolated as a HeLa cells reveals strong staining of nucleoli and weaker nucleoplasmic positive protein complex e.g., staining. with an 82kDa which is not either Immunoblotting experiments also show reactivity of the antisera polypeptide recognized by towards a variety of polypeptides. Production of and monoclonal or WGA and hybridomas mAb501 whose N-terminus has a strong sequence antibodies is currently under way. Our current results indicate that the proteins homology to eukaryotic DNA binding proteins. In summary, the fate that co-purify in these complexes are specific pre-rRNP components, and that of p51 during postmetaphase nuclear formation, and its in vivo the complexes that we have isolated are bona fide pre-rRNP complexes. protein association(s) should be capable of being investigated. Sunday. Proteins of the Nucleus (561-566) 97a 561 562 THE ROLE OF DEPHOSPHORYLATION IN SUB-NUCLEAR THE IDENTIFICATION AND CLONING OFA NOVEL DISTRIBUTION OF pre-mRNA SPLICING FACTORS ((Tom Misteli and David L. Spector)) Cold Spring Harbor NUCLEOLAR PROTEIN NOR-55. ((C.M. WHITEHEAD, R. J. Laboratory, Cold Spring Harbor, NY 11724 WINKFEIN, M.J. FRITZLER, J.B.RATTNER)) Department of Medical Biochemistry, University of Calgary, Calgary, Pre-mRNA splicing factors are distributed non-homogeneously in 20- Alberta Canada. 40 speckles of irregular shape in the mammalian cell nucleus. To investigate the molecular mechanisms involved in the organization of splicing factors in nuclear speckles a semi-intact cell system using We have identified and cloned a novel human nucleolar permeabilized Hela cells reconstituted with nuclear extract was protein designated NOR-55 (Nucleolar Organising Region were and DNA developed. Permeabilized cells active in transcription of 55 This was identified replication when incubated with nuclear extract and dynamic nuclear protein kDa). protein by screening reorganizations, such as rounding up of speckles in response to in- a HeLa cDNA library with a human autoimmune serum. hibition of RNA polymerase II transcription, could be reconstituted in Overlapping clones and 5' RACE products resulted in the permeabilized cells. This redistribution of snRNP- as well as non- isolation of the full length clone. Nucleotide sequence snRNP-proteins was strictly dependent on soluble nuclear factors. By inhibitor titration experiments and sensitivity to Inhibitor-2, ser/thr analysis indicated it encoded a novel protein of a predicted phosphatase I activity was identified to be a key factor in both rounding size of 55 kDa. Antibodies raised to different regions of the up of speckles in response to transcriptional inhibition as well as proper protein all resulted in the same Indirect Immunofluoresence distribution of factors in splicing transcriptionally active, permeabilized NOR-55 is localized to discrete regions cells. These observations suggest a cycle of phosphorylation and pattern. punctate dephosphorylation to be crucial in the organization of pre-mRNA (fibrillar centres) of the nucleolus during interphase. In splicing factors in nuclear speckles. Furthermore, these data provide a mitosis, NOR-55 is present at the nucleolar organising link between the effect of phosphorylation on splicing factor function of chromosomes. In double label IIF and their subnuclear localization. regions metaphase studies we have found that NOR-55 colocalized at all stages of the cell cycle with Upstrearn Binding Factor, NOR-90.

563 564 SUBCELLULAR DISTRIBUTION OF METALLOTHIONEIN AND BINDINGS OF METALLOTHIONEIN TO SUPRANUCLEOSOMAL COPPER IN HUMAN CELLS ((Gonzalez, M., Cambiazo, V., Uauy, R. and FIBERS IN MOUSE PANCREATIC NUCLEI AFTER INDUCTION BY Gitling, J..)) Lab. Cellular & Molecular Biol., Instituto de nutrici6n y 4-AMINOPYRAZOLO [3,4-d] PYRIMIDINE. ((Y. Tohno, S. Tecnologia de los alimentos (INTA). Univ. of Chile., Casilla 138-11, Tohno, T. Minami*, Y. Okazaki*, M. Utsumi, and M.-o. Santiago, Chile. Yamada)) Laboratory of Cell Biology, Department of Organisms as diverse as yeast and mammals share a requirement for the regu- Anatomy, Nara Medical University, Kashihara, Nara lation of cellular copper metabolism, in order to provide appropriate functional 634 and *Faculty of Pharmaceutical Sciences, Kinki structure and catalytic activity of several cuproenzymes. Metallothioneins University, Japan. (MTs) are major intracellular copper/zinc-containing proteins and they act as intracellular stores or buffers for these essential metals. In this work we have To examine intranuclear localization of analyzed relative survival of Caco, Bewo and HepG2 human cells, when chron- metallothionein (MT) induced by administration of 4- ically exposed to different extracellular concentrations of copper. The results aminopyrazolo [3,4-d] pyrimidine (4-APP), the of MT was immuno-electron indicated that HepG2 have a greater capacity of regulating copper excess than present study performed by Bewo and Caco. Depletion of intracellular glutathion (GSH) by diethylmaleate microscopy. One day after oral administration of 4- the mouse removed and frozen with (DEM), under high extracellular copper concentrations, produced a slightly APP, pancreas dry ice was cut at about 1 mm thickness. Pancreatic decreased HepG2 cells survival. In addition, we have examined subcellular distribution of metallothionein in Bewo and cell lines slices were reacted with anti-MT antibody, fixed pattern Caco, HepG2 with and and embedded by indirect inmunofluorescence and Western blots assays, using a monoclonal formaldehyde glutaraldehyde, revealed that MT is both in hydrophilic Lowicryl K4M resin. Ultrathin anti-MT antibody. Inmunostaining pattern, present sections were reacted with the the of cells. This is distributed secondary antibody in nucleus and cytoplasm interphase protein labelled with colloidal and stained both with after 48 hr of to gold, in patches in both compartments. Interestingly, exposition acetate and with lead citrate. It was found cells an enhancement in uranyl copper excess, in presence of DEM, HepG2 displayed that the site was shown in the when to cells incubated in DEM MT simultaneously the inmunofluorescence intensity compared chromatin and in the rough endoplasmic reticulum of was more evident in the cel- or copper alone. This enhancement cytoplasmic mouse exocrine cells one after that in extracellular levels alter the pancreatic day lular domain, suggesting changes copper administration of 4-APP. The structure of MT-binding are in to stablish subcellular distribution of MT. Further studies, progress chromatin was of in the be- composed supranucleosomal fibers, whether this effect correspond to specific modifications relationship but not of nucleosomal fibers. tween subcellular distribution of metallothionein and copper. (Supported by CINUT and Cochilco).

565 566 OF THREE NOVEL FISSION NUCLEAR AND NUCLEOLAR TARGETING OF THE HUMAN r- ISOLATION AND CHARACTERIZATION PROTEIN L7A. ((G. Russo, G. Ricciardelli and C. Pietropaolo)) YEAST CELL-CYCLE MUTANTS THAT ARE RESCUED BY THE SPII Dipartimento di Biochimica e Biotecnologie Mediche, Universit3 di NUCLEAR GTP-BINDING PROTEIN. ((U. W. Mueller, S. S. Salus, D. Ensenat, and S. of Biochemistry, Baylor College of Medicine, Houston, Napoli "Federico II", Via Sergio Pansini, 5 - Napoli, I-80131 Italy. Sazer)) Department TX 77030. The human ribosomal L7a is a of the major protein component The evolutionarily conserved nuclear GTP-binding protein Ran has been We in HeLa cells L7a- ribosomal subunit. transiently expressed implicated in several cellular processes, including cell-cycle progression, mRNA we studied their subcellular betagalactosidase fusion proteins and transport, DNA replication, and nuclear protein import. Ran is converted to its localization by indirect immunofluorescence staining with anti- active GTP-bound state by the chromatin associated protein RCC I and is betagalactosidase antibodies. We have identified three distinct inactivated by the GTPase activating protein RNAI. The downstream effectors of domains responsible for the nuclear targeting of the protein: domain Ran that link the GTPase to its cellular functions have not yet been identified. The I, amino acids 23-51; domain II, amino acids 52-100; domain III, fission yeast homologues of Ran (named spill) and RCC1 (named pimI) have been amino acids 101-220, each of which contains at least one nuclear cloned and conditional mutants in piml have been isolated. These mutants display localization signal (NLS). Through subcellular localization analysis a cell cycle arrest at the mitosis to interphase transition. The pimI mutant cells are of deletion mutants of L7a-betagalactosidase chimaeras, we binucleate, septated, and have fragmented nuclear envelopes. We have isolated three novel conditional mutants that also arrest at the mitosis to interphase demonstrate that domain II plays a special role since it is necessary, of the I GTPase. In addition to although not sufficient, to target the chimaeric betagalactosidase to transition and are rescued by the overexpression spi 1, one of the mutants is also rescued by overexpression of genes involved in the the nucleoli. In fact, we demonstrate that the nucleolar targeting spi proper organization of the cytoskeleton. Analysis of actin structures in this mutant the of domain II an additional basic process requires presence plus reveals a defect in the depolymerization of the actin contractile rings that are an NLS or a basic stretch of domain that can be represented by formed at the site of septation. Thus this mutant identifies a genetic link between amino acids without NLS activity. Thus, when multiple NLS are the spill GTPase and components of the cytoskeleton. Further characterization of functions. Domain II drives present, each NLS exerts distinct these mutants is currently underway and will be reported. nucleolar accumulation of a reporter protein with the cooperative action of a short basic amino acid sequence, suggesting a mechanism requiring protein-protein or protein-nucleic acid interactions. 98a Proteins of the Nucleus (567-572). Sunday

567 568 NUCLEOLAR AND COILED BODY TARGETING OF NOPPI40 AND IDENTIFICATION OF A NEW COILED BODY PROTEIN ((M.C. NAP57 ((C. Isaac and U.T. Meier.)) Department of Anatomy and Alliegro and M.A. Alliegro)) Department of Anatomy, Louisisna State Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461 University Medical Center, New Orleans, LA 70112. Noppl4O and NAP57 are two resident proteins of the nucleolus and the Coiled bodies (CB) are small, round nuclear inclusions that have coiled bodies. Noppt4O constantly shuttles between the nucleolus and the been identified in many somatic cell types. Equivalent structures are cytoplasm. NAP57 is a highly conserved Noppl4O associated protein. Here found in the germinal vesicles of amphibian and insect oocytes, known we analyze the requirements for nucleolar and coiled body targeting of the as and Binnenkorper. Their functions As respectively sphere organelles two proteins employing transient transfection assays. expected, Noppt4O are not known, but their molecular composition is being brought to readily accumulates in the nucleolus, but only subsequently appears in coiled light. In addition to the nucleolar protein, fibrillarin, coiled bodies that either has to be routed the nu- bodies. This suggests Noppl4O through contain an array of RNA processing molecules characteristic of or that levels of i.e. cleolus to the coiled bodies only high expression, longer one that seems to be specifically enriched times, allow accumulation in coiled bodies. These and other pos- spliceosomes. Only protein transfection in CB has been described: We have now identified pigpen, sibilities are presently being tested and the results will be discussed in regard p80-cohin. a new of and Alliegro, to coiled body function. NAP57, contrary to Noppl4O, does not accumulate in member of the EWS family proteins (Alliegro the nucleolus, but is distributed throughout the nucleoplasm. To investigate 1996), as a second protein enriched in CB. In double-label the reasons for this aberrant localization, we determined that (i) the trans- immunofluorescence studies, pigpen colocalized with p80-coilin, but fected NAP57 is not degraded, (ii) the result is independent of the amount of not with the spliceosome-specific protein, SC35. Anti-pigpen expressed NAP57 or the duration of the transfection, (iii) endogenous NAP57 antibodies were subsequently used to study the organization of CB is nucleolar, (iv) the aberrant localization is independent of the HA-epitope during the cell cycle in endothelial cells. In addition to brightly tag, (v) drug induced and reversible dispersion of the nucleolus does not allow staining focal points distributed throughout the nucleus and on the NAP57 to enter the nucleolus, and (vi) co-transfection of Noppl40 does not nuclear envelope, a diffuse nuclear pool of both pigpen and p80-coilin move NAP57 to the nucleolus. Presently we are investigating whether slow was observed in interphase cells. During mitosis, CB undergo a turnover of the endogenous NAP57 prevents the exogenous one from entering period of coalescence in prophase, dispersion in anaphase, and the nucleolus and are also testing individual domains of NAP57 for proper late in the nucleus and perinuclear localization. reassembly during telophase nucleolar cytoplasm. Our results indicate that CB and CB proteins are present in places and at times previously unreported. Whether these findings are reflective of transport and storage processes or specific functional states remains to be determined. Supported by NIH EY09533.

569 570 WHOLE-MOUNT IMMUNOLOCALIZATION OF A MOUSE GERM CELL. IMMUNOFLUORESCENT AND IMMUNOELECTRON MICROSCOPIC NUCLEAR ANTIGEN, GCNA1, IN SPERMATOGENIC CELILS. ((1). Wang LOCALIZATION OF CONFRONTING CISTERNAE. ((J.R. Palisano and J.L. and G.C. Enders)) Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160-7400 Thacker)) Department of Biology, University of the South, Sewanee, TN 37383. of GCNAI is a OkD mouse germ cell nuclear antigen that is recognized by Immunofluorescent and immunoelectron microscopic investigations confront- in the rat monoclonal antibody 10D9G 11. The antigen, of unknown function, ing cistemae (CC) were initiated to determine the origin of CC which are found can first be detected immunocytochemically in mouse primordial germ cells at function of CC I selected mammalian fetal and tumor cells. Although the origin and -l1 days post coitum within sections of the genital ridge. GCNA is remain an electron studies have characterized their struc- continually expressed in oogonia and oocytes until it is lost from dictyate enigma, microscopic oocytes shortly after birth. GCNAI is expressed within mitotically quiescent ture as stacked cistemae that exhibit an intercistemal spacing of 20 nm and an prospermatogonia to haploid spermatids. To characterize more precisely the intracistemal lumen that varies from 20 to 50 nm. The outerfaces of the cistemae distribution of the antigen within the nucleus, a whole-mount light level are always studded with ribosomes, and CC are always devoid of nuclear pores. immunolocalization procedure (Davis, 1993 Methods Enzymol. 225:502-516) Because CC are infrequently observed, several hypotheses concerning the ori- and immunoelectron microscopy were performed on mouse seminiferous of CC that have been at To test the most tubules. In whole-mount preparations, seminiferous tubules were fixed with gin postulated are, best, conjectures. methanol and dimethyl sulfoxide. Interphase spermatogonia appeared weakly prevalent hypothesis that CC are fragments derived from the nuclear envelope stained throughout the nucleoplasm, with prominent specks of heavy staining (NE) of rapidly dividing fetal and tumorcells, an immunofluorescent investigation appearing in varying size and number. Strong staining was associated with of synchronized HeLa cells was initiated using amonoclonal antibodyto lamin B, metaphase spermatogonial chromosomes. During early prophase of meiosis in a NE While 0.5% buffered paraformaldehyde fixations optimized spermatocytes, the antigen appeared in a cord-like distribution with both ends specific protein. attached to the nuclear envelope, following the condensing chromosomes. The immunofluorescent visualization of the NE in nondividing cells,it was not pos- staining became weak within late pachytene spermatocytes. After meiotic sible to detect CC in dividing HeLa cells. The inability to detect CC in HeLa cells divisions, round spermatid nuclear staining was relatively uniform, except for may be the result of CC having lost the lamin B during an earlier stage of NE strong nucleolar staining. Immunogold-electron microscopy on L.R.White disassembly, CC not having originated from NE, or the inability to resolve the embedded sections of periodate-lysine-paraformaldehyde fixed spermatogenic label- cells resulted in localization throughout most nuclei, although not specifically small fragments of CC by immunofluorescence. Preliminary immunogold CC do associated with nuclear organelles, such as the synaptonemal complex and sex ing investigations have localized lamin Bto CC, which would indicate that vesicles or the nuclear envelope. Thus, while whole-mount results suggest that arise from NE since lamin B is a NE specific protein. GCNAl localizes to condensing chromatin or the perichrosomal layer the electron microscopic localization does not provide equivalent, rather equivocal results.

571 572 NUCLEAR PORE GLYCOPROTEIN p62 IS PHOSPHORYLATED BY DYNAMIC ORGANIZATION OF THE PERINUCLEOLAR GLYCOGEN SYNTHASE KINASE 3, A KINASE ENRICHED IN THE COMPARTMENT. ((S. Huang, T.J. Deerinck*, M.H. Ellisman*, and D. L. GLYCOGEN FRACTION OF XENOPUS LAEVIS NUCLEAR Spector)) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; REFORMATION EXTRACTS. and *University of California at San Diego, Department of Neurosciences and ((M R.Caracciolo# B.Fang*i, M.Miller*and J.A.Hanover#)) #Laboratory San Diego Microscopy and Imaging Resource, LaJolla, CA 92093. and Biology,NIDDK, NIH, Bethesda MD of Cell Biochemistry The structure preferentially of Biology Wright State UniversityDayton, Ohio perinucleolar compartment (PNC) is a nuclear *Department localized at the periphery of the nucleolus. Several small RNAs transcribed by iCo-presenter RNA polymerase III (i.e. MRP RNA and RNAse P Hi RNA) and the polypyrimidine tract binding protein (PTB, hnRNP I) have been identified in A family of nucleoporins have been described which are modified by 0- the PNC (Ghetti, et al., 1992. Nucleic Acids Res. 20: 3671-3678; Matera, et linked N-acetylglucosamine and SerIThr phosphate. Evidence also points al., 1995. J. Cell Biol. 129: 1181-1193). However, the structural and these nucleoporins. These modifications are to sialylation of some of functional characteristics of the structure have not been extensively studied. In in Xenopus laevis nuclear reconstitution extracts dynamic. When examined this we have further characterized the structural features of the PNC. in of phosphorylation increases dramatically during mitosis. report, vitro, the level with a monoclonal specifically recognizing are involved in nucleoporin Using immunostaining antibody To examine which enzymes phosphorylation, we have examined the structure of the PNC at high resolution by of kinases for their ability to phosphorylate PTB (SH54), we tested a number electron We have found that the PNC is in direct contact with the recombinant nucleoporin p62. Although p62 was not modified by Protein microscopy. surface of the nucleolus. To visualize the PNC in living cells, we have kinase A, protein kinase C, or casein kinase, glycogen synthase kinase-3 constructed a fusion protein between PTB and green fluorescent protein. We (GSK-3) did phosphorylate p62. The substrate specificity of GSK-3 is of PNCs GlcNAc transferase which glycosylates have performed time-lapse studies and have found that the number very similar to that of the O-linked nucleus to be but the size and shape of the PNC is dynamic that the same or adjacent sites might be modified by both per appears stable, p62 suggesting over time. In addition, we have also found that the number of PNCs in the enzymes. To determine whether GSK-3 was present in Xenopus extracts suggesting that the and found that GSK-3 is selectively enriched nucleus is inherited from parental cells to daughter cells we assayed various fractions PNCs are mitosis. The PNC during mitosis and previously shown to be required for nuclear segregated during dissociates in a glycogen-rich fraction reforms at late in the nuclei prior to the reentry into the are currently exporing the relationship between 0- telophase daughter reformation in vitro. We of a large possible sialylation, and GSK-3 mediated nuclei of the majority of PTB. Interestingly, through examination linked GlcNAc addition, number cancer that PNCs are of the nucleoporins. of human and normal cells, we have found phosphorylation predominantly present in cancer cells and are present in a significantly lower percentage of normal cells. These findings suggest a potential relationship between the PNC and carcinogenesis. Sunday. Proteins of the Nucleus (573-578) 99a

573 574 ANALYSIS OF NUCLEAR LOCALIZATION OF LBP/P40. ((M. Sato, K. TEMPERATURE SENSITIVE ALLELES OF NOP2 ((B. Hong, P. Wu, J. Kinoshita, Y. Kaneda, Y. Saeki, A. Iwamatsu+, K. Tanaka and Y. Kaneda.)) S. Brockenbrough, and J. P. Aris.)) Department of Anatomy and Cell I.M.C.B. Osaka Univ. Osaka, Japan. and +Central Laboratories for Key Biology, College of Medicine, University of Florida, Gainesville, FL 32610. Technology, Kirin Brewery Co., Ltd. Kanagawa, Japan. E-mail: johnaris~lcollege.med.ufl.edu. We isolated monoclonal antibody M108, which recognized the 40 kDa protein NOP2 encodes an essential nucleolar protein in yeast. We have previously (p40). The p40 localized on perichromosomal region in mitosis, on perinuclear shown that Nop2p plays a role in ribosome synthesis and is specifically re- region in interphase and on protein particle in the cytoplasm. The morpho- quired for normal processing of the 27S pre-rRNA to 25S rRNA found in the logical observations and biochemical analyses indicated that the antigen was 60S subunit. Protein sequence homology comparisons, and RNase protection associated with the nuclear envelope and chromatin DNA, thus, it was con- experiments suggest that Nop2p may function as an rRNA methyltransferase, sidered to be a candidate for the ligand protein between the nuclear envelope and other experiments are underway to explore this possibility further. To and chromatin DNA. We purified cytoplasmic p40 by FPLC, and peptide explore potential interactions between Nop2p and other yeast proteins, we sequencing showed that cytoplasmic p40 was identified with LBP/p40, which have used both the two-hybrid system of Fields and colleagues, and the two- was known as a precursor of laminin binding protein p67 (LBP/p67). As there hybrid system as modified by Elledge and coworkers. However, this effort has was no report to reveal nuclear localization of LBP/p40, next, we confirmed yielded only negative results. To pursue genetic approaches to Nop2p func- whether nuclear p40 was LBP/p40 or not. We obtained LBP/p40 cDNA tion, we have generated conditional lethal nop2 alleles. Six different alleles from HeLa cDNA library by PCR method. The c-myc tagged LBP/p40 gene have been isolated and sequenced, and all are characterized by multiple amino was transfected with mammalian cultured cells, and immunofluorescence was acid replacements and by deficiencies in 60S subunit synthesis at 37C. One is carried out using anti-c-myc tag antibody as a probe. Besides cytoplasmic sensitive to formamide at permissive temperature in addition to being tem- reticular signals, nuclear localization of c-myc tagged LBP/p40 protein was perature sensitive in the absence of formamide. Subsets of, or single, amino observed in transfected cells. Nuclear fractionation of stable transformant of acid substitutions are being tested for their ability to confer the ts (or fs) T7 tagged LBP/p40 demonstrated that high salt-detergent treatment was re- phenotype. Screens for high copy number suppression and mutations that are quired for isolation of nuclear LBP/p40, and either DNase I or 150 mM salt synthetically lethal with nop2 alleles are underway. treatment did not work. These results revealed that LBP/p40 protein local- ized not only on ribosomes in cytoplasm as reported by several groups, but also in nucleus with tight association with nuclear structure.

575 576 BX34: A LARGE, FILAMENTOUS PROTEIN LOCALIZED TO THE PURIFICATION AND LOCALIZATION OF DROSOPHILA DMR EXTRACHROMOSOMAL CHANNEL NETWORK AND NUCLEAR PROTEIN ((K. Kawasaki', E. Akaboshi2 and T. Shibata'.)) 'Laboratory of PORE COMPLEXES IN DROSOPHILA ((G. Zimowskal"2, J.P. Aris2, H. Cellular and Molecular Biology, The Institute of Physical and Chemical Saumweber3, and M.R. Paddy12.)) 'Center for Structural Biology and Research (RIKEN), Wako-shi, Saitama 351-01, Japan, 2Institute for 2Department of Anatomy & Cell Biology, University of Florida, and Molecular and Cellular Biology, Osaka University, 1-3 Yamanaka, Suita 565, 3Biologische Institute, Abt. Zytogenetik, Humboldt Universitaet zu Berlin. Japan. We have previously described the Drosophila Bx34 protein as a large (262 DMR cDNA isolated from Drosophila melanogaster was homologous to the kD), predicted filamentous protein localized to the nuclear interior and the yeast DMC1 and RAD51, which are known to play crucial roles during meiosis nuclear periphery which showed moderate homology to the human/rat Tpr and during both meiosis and mitosis, respectively, and whose gene products are nuclear pore complex protein over its entire length. Over the last year, it homologous to each other and to the RecA protein of Escherichia coli(Akaboshi has become clear that Bx34 in the nuclear periphery localizes to nuclear pore et.al. (1994) Jpn. J. Genet. 69 663-370). To analyze localization and in vitro complexes. Furthermore, we now have strong evidence for the internal nuclear activity of DMR protein, we expressed the DMR protein in Escherichia coli localization of Bx34 from immunofluorescence staining of thin physical sec- and prepared affinity-purified antibody against DMR protein. Immunoblot tions from giant salivary gland polytene nuclei. Staining with either the origi- analysis showed that the DMR protein existed in ovary, early embryos and nal Bx34 monoclonals or affinity-purified polyclonals raised against a peptide few in adults. We purified the DMR protein by immuno-detection with the from predicted Bx34 sequence produces an indistinguishable staining pattern. antibody from Escherichia coli in which DMR protein was co-expressed with In the nuclear interior, Bx34 stains extra-chromosomal and extra-nucleolar thioredoxin. The DMR protein was elated in a void volume in gel filtration, regions (the "extrachromosomal channel network") with a sub-structure that suggesting that the protein caused self-polymerization as RecA protein does. often appears filamentous. Several natural speculations arise for the function The DMR protein binds single-stranded DNA and double-stranded DNA. The of a filamentous protein wh ich is localized from the edges of chromosomes chromatogaphic behavior and biochemical property of DMR protein were sim- and the nucleolus to nuclear pore complexes, not the least of which is a role ilar to those of the RecA protein of Escherichia coli, but not identical. These in nucleoplasmic transport. We are currently testing Bx34 function using a data suggested that DMR protein would be involved in DNA metabolism, such combination of P element-mediated mutagenesis, antibody injection into early as recombination and/or repair, in meiosis as well as in mitosis. Drosophila embryos, and immunofluorescence co-localization studies of Bx34 and components of pre-mRNA metabolism.

577 578 INCREASED RIBONUCLEOTIDE REDUCTASE ACTIVITY IN NUCLEAR RECONSTITUTION IS INDEPENDENT OF THE LENGTH OF DNA HYDROXYUREA-RESISTANT MOSQUITO CELLS. (( K.M. Shih FRAGMENTS IN CELL-FREE EXTRACTS OF XENOPUS L4EVJS EGGS ((Z.F.Jiang and Z.H.Zhai)) College of Life Science, Peking University, Beijing, and A.M. Fallon)) Departmentof Entomoogy, University of Minnesota, China,100871 St. Paul, MN 55108.

The details on how exogenous DNA reconstitutes nuclear structure in cell-free Hydroxyurea-resistant A adsa Wpicusmosquitocellswer selected extracts from activated eggs of Xenopus Iaevis are not yet clear. By digesting pBR322 by incrementalexposure ofunm edcellsto hydroxyurea with mixed restriction endonucleases, we have prepared DNA fragments with lengths of and and them to the egg extracts respectively. It is found that corntrationsrangingfrom 0.1 to8 mM. Cbnal populations that had 750bp. 375bp 186bp, add become 40fold moreresistant to droxyureaman wild typecells varied even the 186bp DNA fragments can assembly many typical nuclei which are surrounded in morphology, andtheirgrowth ratedecreased to an approximate 45 h by double membranes with nuclear pores in this cell-free extracts. Microspectrophotometry demonstrates that DNA fragments undergo obvious changes doublng time, relativetoan 18 h doubling time in unselected cells. At from condensation to decondensation during nuclear reconstitution, while electrophoresis r a this level of resistance, th cells neddpoid, with modal shows that DNA fragments have not been linked with eath other after reconstitution. chromosonenumberof 6. When bedwith S(methonicysteineI, The reconstituted nuclei are capable of DNA replication and the activity is positively clone HU 1062, whichgrew n the prese of 8 mM hydroxyurea, related to the length of DNA fragments in a given extent. It seems that the nuclear overproduceda ledprotein wih the approximate szeof the45,000 reconstitution in Xenopus cell-free extracts is independent of not only the resources of Dalnon M subunitof ribonucleoade reductase. Consistentwith this exogenous DNA, but also the length of DNA. observation, rlbonucleotide reductase activity in HU-1062 c6Ilswas roughly 10-fold higher than in wild type control cells. This is thefirst exampleot an hydroxyurea-resistant insect cel line. lOOa Chromatin and Chromosomes I (579-584). Sunday 579 580 TIE EXACT LOCALIZATION OF SCEPOItTS ON THE CaHOMOSOME 1 DECONDENSATION OF HETEROCHROMATIC CHROMOSOME REGION FOR HEALTHY CARRIERS WITH HBsAg POSiTIVE AND THIR NEWBORN INDUCED BY VP16 ACIDIC ACTIVATION DOMAIN. ((T. Tumbar, G. Li, CHtOREN. ((YIng 0)) Departnent ofCalnical Genetics, Oingdao Second G. Sudlow, and A.S. Belmont)), Department of Cell and Structural Biology, People's Hospital, Qingdao, 266033, P.R. CAia. (Spon. by DoiJxA.) University of Illinois, Urbana-Champaign, Urbana, IL 61801

The previous studies with analysurg for sister chrometid exchange (SCE) We have examined changes in large-scale chromatin structure induced by frequenes in peripheral lymhocytes lid slowed a significant increase targeting the acidic tail of the transcriptional activator VP16 to specific from hepatitis B patients with HBsAg positive compared to controls. The chromosomal regions. Previously we described the interphase structure of a pinpose of this study was to do the hirther research in cytogenetics for late replicating, heterochromatic chromosome region, produced by gene the healthy cares with MsAg positive and their newborn children from amplification of a transfected DHFR gene (Li, et al., Mol. Biol. Cell. 6 sup.: a new point of view. The object of suAy was a total of 24 invdias. 71a, 1995) with an adjacent 256 copy array of lac operators. This 6 healthy cars with HBsAg positive and 6 of theyr newborn children homogeneously stained region (HSR), is - 80100 Mbp in size and through were observed. 8 normal adidts and 4 of their newborn children were most of the cell cycle exists as a condensed mass adjacent to the nuclear ansiysed for coparson. The reason that choosing newborn was to exclude envelope with comparable compaction to its metaphase conformation. After infectious and environment factors. The method to be used was the tech-- transient expression of a lac repressor VPl6 acidic activation domain fusion neqw of complex display for SCE and G bands, it was groped by ourselves. protein, a striking decondensation of the large-scale chromatin structure was We observed and recorded the every position of SCE points on te chromo- observed; the majority of nuclei showed an uncoiling of the original condensed, some 1, the exact sites were ocid at the regions and bands. The -.75 um diameter HSR into long, extended fibers occupying a large fraction of subjects were divided into 7 groups accorckug to sex, age, fHsAg positive the nuclear volume. In nuclei with highly extended fibers, measurement of the or negative and etc. There was no significant difference between d total length of fibrillar staining, 40 um, indicated a -1000:1 compaction ratio. cam and control groups in either the SCE frecjency or the distrbution In 10-25% of transfected cells, extended fibers of the HSR were coiled on the of SCE on the chromosone 1 (P>0.06). We next plan to investigate into surface of a spherical volume - 1-3 um in diameter, devoid of internal DAPI other pars of 22 chromosomes, compete Nis research, and reveal father staining. In addition to the large-scale chromatin decondensation induced by the the mechanism of hepatitis B at the cytogenetic level. VP16 activation domain, a movement of the HSR away from the nuclear envelope into the nuclear interior was observed. Preliminary results indicate this HSR is composed of gene amplification units containing -15 copies of the transfected vector separated by - 500kb flanking DNA segments. Our results suggest therefore that targeting high levels of the VP16 activation domain leads to an uncoiling of heterochromatin and decondensation of large-scale chromatin structure which propagates over large DNA lengths.

581 582 TWO TYPES OF DNA LOOPS IN INTERPHASE CHROMOSOMES UPREGULATION OF NEUROFILAMENT LIGHT CHAIN IN PC12 (M.V. Glazko-., N.N. Kireeva, A. Kleimans) CELLS CORRELATES TEMPORALLY WITH MIGRATION OF Institute of General Genetics, Russian Academy of Sciences, Moscow 1 17809, Russia DNAse HYPERSENSITIVE CHROMATIN TO THE NUCLEAR PERIPHERY. ((P.C. Park and U. De Boni)) Department of Interphase chromosomes are organized as a series of topologically independent of M5S 1A8 chromatin loops. We have shown previosly that interphase chromosomes have two Physiology, University Toronto, Toronto, ON, types of loops. One type of loops (up to 200-300 kb) is determined by the interaction of The contents of interphase nuclei is organized in a non-random interphase chromosomes with the nuclear envelope. Other DNA loops (up to 5 kb) manner. To test if this structural organization represents functional form rosette-like structures which are the form of organization of tissue-specific compartmentalization of nuclear events, we examined the relative transcriptionally inactive genes. spatial distributions snRNPs, and of DNAse Hypersensitive We analyzed in vitro the specificity of DNA-protein interaction in the course of Chromatin (DHC). This was carried out in nuclei of PC12 cells formation of rosette-like structures of interphase chromosomes and upon the during Nerve Growth Factor (NGF) induced differentiation. DHC and association of interphase chromosomes with the nuclear envelope. To this end, we used snRNPs extensively colocalized as pan-nuclear speckles in untreated cloned DNA fragments of mouse interphase chromosomes isolated from the protein controls. Upon NGF exposure, both migrated to the nuclear periphery cores of rosettes and from nuclear envelopes of hepatocytes. A high specificity of both to form a shell apposed to the inner nuclear membrane. The types of DNA-protein interactions was observed in the case of double-stranded DNA migration of DHC proceeded that of the snRNPs resulting in their fragments. It has also been found that single-stranded DNA fragments interact with transient, spatial separation prior to re-establishment of colocalization a low specificity with the protein cores of rosettes and with a high specificity with at the periphery. To address whether snRNP nuclear Differences in the specificity speckles represent envelopes. of binding of circular supercoiled functional compartments where splicing occurs, or a storage and relaxed molecules of plasmid DNA to the protein cores of rosettes and to pool, the the expression of neurofilament light chain (NF-L) gene was nuclear envelope have also been revealed. quantitatively assessed by in situ hybridization. The sigmoidal time Proteins specifically interacting with various "structural" regions of interphase course of NGF-induced upregulation of NF-L closely resembled that chromosomes have been identified. of the formation of DHC shells, but not of snRNP shells. We The results obtained suggest a high importance of the conformational state of conclude that the expression of NF-L in PC12 cells is associated chromosomal DNA regions in the formation of two types of loop structures in with changes in the position of the gene sequence relative to the interphase chromosomes. nuclear periphery, rather than its association with snRNP foci.

584 ORDER IN MITOTIC HUMAN AND MOUSE RADIATION-INDUCED CHROMOSOME DOMAIN REARRANGEMENT IN ((T. Freeman, Gu, J Fazekas, K. Lee H. Lee and R. Nagele)) HUMAN FIBROBLASTS. ((D.R. Boreham, J-A Walker, D.L. Brown*, and Department Biology, University of Medicine and Dentistry of R.E.J. Mitchel)) AECL, Chalk River Laboratories, Chalk River, Ontario,

NJ 08084 and Canada, KOJ Jersey, Stratford, Department of Biology, Rutgers 1J0, *University of Ottawa, Ottawa,Ontario, Canada, KIN 6N5 University, Camden, Chromosomes are located within the interphase nucleus in regions or territories called domains. Using FISH, a pair of homologous chromosomes have been mapped to precise positions on the chromosomes can be visualized as two discrete domains. The spatial prometaphase chromosome rosette in human fibroblast cell lines with location of the two homologous chromosome domains may be influenced by hybridization (FISH), suggesting that this chromosome factors such as cell cycle, orientation of the nucleus, level of gene activity, arrangement type-independent and species-specific (Science, 1995, DNA and chromatin damage, etc. We have examined the spatial location 270:1831). objectives of the present study were to resolve the spatial of chromosome 21 domains in attached confluent human fibroblast cells rosettes of normal human cells and determine if using confocal microscopy. Cell nuclei were scanned with a miniumof five precise chromosome spatial ordering exists in other species. Chromosome 0.5 pm sections and the composite image was analyzed. The distance spatial predicted using FISH mapping data, statistical analysis between the two chromosome 21 domains was calculated for each nucleus. associations metaphase preparations, and Bennett's method Our results demonstrated that in control cells or in cells examined immediately after gamma-rays), (Chromosomes Today, 1984, 8:190) for chromosome positioning based on irradiation (4 Gy, 66.2±2.1% of chromosome

chromosome 21 domains were greaterthan 4pm apart (33.8±1.9% were length. The predicted order was tested using FISH together). However, when cells were examined two hours after a 4 Gy exposure, chromosome-specific probes. Results support the idea that each only 30.5±2.1% of the chromosome 21 domains were greaterthan 4 pm apart has precise location on the rosette at mitosis. A strong (69.5±2.1% were together). Analysis of the domain distance distributions relationship known frequency of specific chromosomal showed that radiation caused a significant rearrangement of chromosome 21 proximity of participant chromosomes on the rosette domains. We propose that domains of homologous chromosomes rearrange observed. Mouse cells were also found to exhibit rosettes at mitosis within irradiated nuclei to facilitate repair of damaged DNA. and, cells, homologs are positioned on opposite sides, suggesting spatial organization may be a common feature Supported by the CANDU Owners Group cells. [Supported by New Jersey Cancer Commission]. Sunday. Chromatin and Chromosomes I (585-590) lOla 585 586 UNUSUAL KARYOTYPES AND TRANSLOCATIONS IN Physalaemus RESTRICTION rDNA ANALYSIS OF THREE MUSCOID FLIES. ((L.V.V. Hode', petersi (AMPHIBIA, ANURA). ((L.B. Lourengol, S.M. Recco-Pimentel', AJ. S.M.Recco-Pimentel', R.F. Bergamo2 and R.M.P. Avancini2)) Department of Cell Cardoso2)) 'Dept. Cell Biology and 2Dept. of Zoology State University of Biology' and Department of Parasitology2, lB, UNICAMP, 13083-970, Campinas, Campinas, CP 6109, 13083-970, Campinas, SP, Brazil. (Spon. by S.M. Oliani) S. P., Brazil Three species of flies (Haematobia irritans, Muscina stabulans, and Chrysomya Sixty two specimens from two populations of Physalaemus petersi putoria) which present economic and sanitary importance had their rDNA studied from the Brazilian West Amazonian were studied. Chromosomal preparations and compared. Genomic DNA was diested with the restriction enzymes: Eco RI, were obtained from cellular suspensions of intestinal epithelium and testis. C- Bam Hi, Hind lIl, Pst I, Sal I, Bgl II and Sma I. Probing DNA from each species with banding and Ag-NOR techniques were carried out for both tissue samples. All the recombinants plasmids pDm 238, HM 123, HM 456 and pcHi 18, we identified some a ones on the animals studied showed 2n=22, but two distinct karyotypes were detected conserved sites and few unique each rDNA. The conserved sites among the three studied species plus two species from literature, Drosophila among the specimens from one of the populations analysed. The unusual melanogaster and Calliphore eryfrocephala, were: Eco RI, Hind lIl, Bgl II and Sma karyotype was observed in three males whose calls differ from the other I located in the coding region 18S, and Pst in the internal transcribed region (ITS). individuals of the same population, suggesting a re-evaluation of the No Bam Hi site was identified in any of the three rDNA. M. stabulans and C. Physalaemus peters taxon. This unusual karyotype presents a heteromorphic putoona presented the same number of sites Hind lIl, Bgl II and Sma in the coding pair of chromosomes but the relation of this pair with sex determination could region 28S but they mapped in different regions. Furthermore, C. putona was the not be elucidated because no female was analysed. The other karyological only fly presenting a second Eco RI site located in the intergenic region (IGS). C. putona had the longest rDNA cistron which was estimated to be 11Kb, when group showed a XXS/XYol chromosomal system, C-band heterochromatin and compared to H. inrtans (9.8Kb) and to M. stabulans (9.0Kb). Restriction maps of NORs polymorphisms. Bivalent chromosomes with a heterozigous C-band the rDNA cistron in C. putona and the other calliphorid, C. erytrocephala, are more frequently showed a lack of chiasma in the region of this heterochromatin similar than maps of the two muscid, H. irritans and M. stabulens. A number of during the first division of meiosis. Multivalent configurations were observed variant bands detected in some digestions may be explained by the presence of in meiotic prophase I of both karyological groups, which indicates the insertions in the 28S region as well as intraspecific variation in the IGS length as occurrence of Robertsonian translocation in these animals. These results described in other Diptera. Species-specific patterns were identified for the three flies when the DNA was digested with each of the following restriction enzymes suggest a high karyological evolution rate and agree with the theory relating Eco RI, Bgl II and Hind lIl (probed with pDm 238). the life history strategies that encourage inbreeding and the karyological SUPPORTED BY: CNPq, CAPES, FAEP evolution of tropical frogs. Supported by CNPq

587 588 RETINOID INDUCED ALTERATION OF THE CHROMATIN STUCTURE IN ELUCIDATING THE STRUCTURAL ORGANIZATION OF THE MAIZE THE RARB PROMOTER. ((Nisan Bhattacharyya, Anup Dey, Saverio GENOME. ((Anna-Usa Paul, Robert J. Fed)) Department of Horticultural Minucci, Sam John*, Gordon HagerA and Keiko Ozato)) Laboratory of Sciences, University of Florida, Gainesville, FL 32611 Molecular Growth and Regulation, NICHD, NIH, *Tularik Inc., South San Francisco, CA 94080 and ALaboratory of Molecular Virology, NCI, NIH. The eukaryotic genome Is organized Into topologically Independent looped (Spon.by Dr. Keiko Ozato) domains that are created through attachments of the chromatin fiber to the nuclear scaffold. Loop size varies among organisms, and usually ranges from P19 embryonal carcinoma cells respond to reinoids and undergo 5kb to 200kb. The gentle lysis of maize nuclei Imbedded In agarose generates a light bend on CHEF (pulsed field) gels that migrates to a position greater than differentiation. One of immediate early genes induced by retinoids is the 700kb. It has been demonstrated In animal systems that that the base of the loop gene. The promoter contains the canonical RARE to which RARP RARP (where the chromatin fiber is attached to the protein scaffold) can be cleaved the RAR/RXR heterodimers bind. We have studied retinoid mediated through the action of the cytotoxic drug VM2S and by use of the changes in the chromatin structure for the stably integrated muticopy RARi judicious endonuclease DNase I. promoter connected to the luciferase gene. This promoter was activated The base of a loop appears to be more sensitive to DNase digestion than upon retinoid addition in P19 cells. We have also studied chromatin the surrounding chromatin fiber. United In situ digestion of maize nuclei with changes in the endogenous single copy RARI2 promoter. We show that DNase results In the conversion of the >700kb genome band to an retinoid treatment induces restriction site hypersensitivity near the RARE, accumulation of fragments ranging from 20kb to 80kb, thus, the maize genome which can be observed both for the transfected and endogenous appears to be organized into domains that fall within this range. Specific promoters. This hypersensitivity was associated with the in vivo promoter hybridization with the maize Adhl gene illuminates a broad band spanning the occupancy in the stable clone. An RAR selective ligand TTNPB, capable of 30-40kb range plus a tighter band of about 75kb. activating the promoter induced hypersensitivity, while an RXR selective VM26 creates a cleavable complex with topolsomerase 11 (an Integral ligand SR11237, failed to induce hypersensitivity. Interestingly, an RA component of the nuclear scaffold and Scaffold Attachment Regions). The antagonist Ro 41-5253 that inhibits RA induced promoter activation also treatment of maize nuclei with VM26 also creates two sets of hybridizing inhibited RA induced restriction hypersensitivity. This hypersensitivity was domains, a broad set falling between 30kb and 45kb which corresponds to the lost upon withdrawal of retinoids from the medium indicating that RA location of Adhi in the DNase treatments, and a tighter band of about 18kb that induced hypersensitivity is closely coupled with the binding of liganded is unique to the VM26 treatments. RARIRXR heterodimers to the RARE. We have also investigated sensitivity The fact that we see distinctly hybridizing bands indicates that the genome Is to micrococcal nuclease and DNasel digestions. Our results suggest that not randomly distributed with respect to the location ofIndividual genes. We are currently attempting to map the relative position maize retinoids induce an alteration in the local chromatin structure that occurs of Adhl and other genes within these looped domains and we are concomitant with transcriptional activation of the gene. evaluating the effects of specific gene activation on the organization of the topological loop.

589 590 NON CATALYTIC ROLE OF DNA TOPOISOMERASE II IN CHROMOSOME Novel Activity of Topoisomerase I ((T. T. Wu.)) Northwestern University CONDENSATION AND IN THE CONTROL OF CHROMATIN TOPOLOGY Topisomerase I normally relaxes supercoiled DNA molecules in a buffer ((K. Bojanowski, A.J. Maniotis, S. Plisov*, A.K. Larsen,* and D.E.Ingber)) containing 50 mM Tris-HCl pH 7.5, 0.1 mM EDTA, mM DTT, 20% glycerol Departments of Surgery and Pathology, Children's Hospital and Harvard Medical and 50 mM NaCl, at 37 C for 30 min. This is achieved by cutting one DNA School, Boston, MA 02115; of Structural Biology and *Department Pharmacology, strand, relaxing and resealing the DNA strand without leaving a trace. It is CNRS URA 147 Institut Gustave Roussy, F-94805 Villejuff, France thus known as the "magicians' magician". We have recently noticed another activity of topoisomerase I DNA topoisomerase11 (topo11) plays an essential role in chromatin condensation (from Promega). When supercoiled DNA molecules and chromosome assembly through a mechanism which is so far poorly are purified in the presence of mRNA attaching to one of its two strands, understood. A major unanswered question is whether the chromatin condensing these intact circular molecules can separate into two distinct bands on agarose activity of topo 11 is dependent on its catalytic activity. Here, we address this gel electrophoresis in TAE buffer adjusted to pH 8.5 with low current and question using a model system based on micromanipulation, which enables the long run-time. These two bands most likely represent the two complementary analysis of the mechanism of chromatin decondensation and recondensation within single-stranded DNA circles. On treating these supercoiled DNA molecules intact chromosomes under defined conditions, in vitro. In this model, continuous with topoisomerase I in a buffer containing 12.5 mM Tris-HC1 pH 7.5, 1 mM chains of mitotic chromosomes are microsurgically removed from living cells and EDTA, 0.05 mM DTT, 2.5% glycerol and 2.5mM NaCl, most of which are from decondensed by heparin treatment or by limited proteolytic digestion. Incubation the topoisomerase I storage buffer, at 37 C for 30 min., only one band appears of proteolyzed or heparin-treated chromosomes with purified topo 11 resulted in on agarose gel electrophoresis under the same condition. That band moves recondensation within minutes and nearly complete recovery of original slightly faster two chromosome shape, size and distribution. This effect was dose-dependent, than the bands mentioned above. The major difference saturable, and controlledstoichiometrically, rather than kinetically. The possibility between these two reaction buffers is the amount of glycerol. We suspect that that topo11 induces chromosome condensation through binding to DNA rather than this novel activity of topoisomerase I may be the physiological one, since the its enzymatic modification was also supported by the finding that recondensation glycerol concerntration is much lower. Furthermore, its effect of holding the was observed in the absence of ATP and in the presence of amsacrine, a potent two bands together can be reversed in the presence of 80 mM magnesium inhibitor of topo11 catalytic activity. Chromosome-bound topo11 retained the ability to chloride, suggesting that topoisomerase I may play an important role in the physically interact with DNA since incubation of topo Il-reconstituted chromosomes long-range effect of maintaining the separability pf the two complementary with a non-hydrolysable ,yimido analog of ATP, (AMP-PNP) was found to stabilize single-stranded DNA circles in intact circular dsDNA supercoils. their structure and prevent their decondensation in the presence of high salt. These results show that in our system topo11 can act directly to drive chromosome condensation and that this effect results from direct protein binding interactions, rather than from this enzymes catalytic activity 102a Chromatin and Chromosomes I (591-596). Sunday

591 592 Investigation of Deoxyribonucleases by the monoclonal against Mn-DNase. Preparation to cryptobiosis in ciliates is accompanied by passage of somatic

((M.M.Belova, L.Y.Machmutova, E.Y.Kotlyr , A.N.Askarova, I.A.Solovieva, nucleus chromatin to liquid-crystalline state. ((A.A. Samoshkin, V.G.Vinter.)) Kazan State University G.I.Sergejeva, N.N.Bobyleva, M.Da Conceicao, F.Livolant.)) Institute of We are presenting the technique of monoclonal antibodies against Mn- Cytology, St.Petersburg 194064, Russia; University Paris-Sud, Orsay 91405, dependent DNase from rat liver chromatin. We have also characterized the France. interactions of antibodies and various nucleases Mn- DNase was purified from As was reported elsewhere, the somatic nucleus (macronucleus - Ma) of rat liver nuclei. To obtain hybridomes myeloma cells (line SP2/0-Ag and line two Bursaria species (Ciliophora, Protozoa) may be used as a good model NS/1-Ag4) were mixed with mouse spleen B-lymphocytes that produced the system for studying ultrastructural and supramoleclular chromatin (Chr) or- antibodies against Mn-DNase from the chromatin in titre 1:64. 11 monoclones ganization. Our study of precryptobiotic (encysting) cells and cryptobiotic that produced antibodies were selected by clonning and recloning and were (resting cysts) of these ciliates by methods of thin sections, Chr spreading used to ascyte production. Antibodies were selected from ascytes by affin- and freeze-fracture revealed drastic changes in Chr supramolecular organi- ity chromatography. Homogenicity of purified antibodies was confirmed by zation as compared with vegetative active cells. A large part of Chr lost electrophoresis. Selected antibodies inhibited the activity of Mn- DNase to their clumping nature and loop architecture to be eventually condensed in 56 percentages. Using immunoenzyme analysis it has been established that rod-like chromonemes, the majority of which looks as a variety of polygonal monoclonal antibodies effectively bind to DNase from Misgarus fossilis embry- structures on the fine sections and spread Chr preparations. Use of freeze- onucleus, Ca,Mg-dependent DNase from Strongylocentrotus intermedius and fracture methods revealed such a strong compactization of cyst Chr, which RNase of Bacillus intermedius. Cross reactions with bovine pancreatic DNase, makes impossible to recognize any karyoplasm components. The arrangement RNase and nuclease of Serratia marcescens were not observed. The obtained of intranuclear fibers in karyoplasm strongly suggests their liquid-crystalline data give evidence that antigen determinant, against which monoclonal anti- organization. Chr of new formed and aged cysts may differ in proportion bodies were selected, exist in nucleases from different organisms and performs, of ordered structures, their inner organization and density. Use of electron probably, common life function. autoradiography combined with method of Chr spreading showed the pres- ence of DNA in both unordered nuclear structures and polygonal plates. Our data provide a new evidence on drastic changes in organization of Chr that occur during preparation to cryptobiosis and at cryptobiosis proper, and can be regarded in relation to some hypotesis dealing with the functional role of condensed DNA and Chr.

593 594 VISUALIZATION OF THE TRANSCRIPTION/REPAIR XPB/ERCC3 DOUBLE LABELLING OF REPLICATING DNA COMBINED WITH PROTEIN FOCI AND REPLICATION/REPAIR PCNA PROTEIN FOCI FISH TO INVESTIGATE THE ORGANISATION OF THE INTERPHASE IN UV-IRRADIATED HUMAN CELLS. ((Solovjeva L.V.,Svetlova M.P. and CELL NUCLEUS ((A.E. Visserl, R. Eils2, H. Bornfleth2, A. Jauch2, P.J.M. Tomilin N.V.)) Institute of Cytology, Russian Acad. Sci.,St-Petersburg Bakkerl, T. Cremer2, J.A. Atenl.)) 1) AMC, Amsterdam, The Netherlands Helicase encoded by XPB/ERCC3 gene is a component of well known tran- 2) University of Heidelberg, Germany scription initiation factor TFIIH and serves both in opening up the DNA in A new approach, combining double labelling of replicating DNA with FISH, promoter regions and in nucleotide excision repair. Using specific anti-XPB was developed to study the 3D localisation of DNA replication within inter- antibodies it was shown that Triton X-100 extracts the majority of the protein phase chromosome territories. Asynchronous primary human female fibrob- from the nuclei but UV-irradiation induces non-extractable DNA-associated lasts were first labelled with IdUrd, chased few hours in normal medium and XPB-complexes which possibly mark the sites of DNA repair. This conclu- then labelled with a second label, CldUrd. After the second label the cells sion is confirmed by the absence of XPB-induction in repair-deficient cells were fixed and hybridised with either a consensus alpha-satellite probe, de- of Xeroderma pigmentosum (XPA complementation group) where UV-lesions tecting centromeric regions, or a X chromosome library. The three labels were cannot be eliminated. The accessory protein of DNA-polymerases delta and detected in different colours by immunocytochemistry. Nuclei containing all epsilon, PCNA, in Triton X-100-treated cells is observed in the focal sites of three colours were scanned using a Confocal Laser Scanning Microscope, and DNA replication and is completely extracted from non-S-phase cells. After the localisation of the labels was analysed by image processing. Centromeres, UV-irradiation non-extractable PCNA- complexes are induced in non-S-phase which are known to replicate later in S-phase, colocalised mostly with the late both repair-proficient and deficient cells. This phenomenon indicates that replication label (CldUrd) and not with the early label (IdUrd). The territory PCNA may play a multiple role in post- irradiation processes: it participates of the inactive X chromosome (Xi) was identified by Barr Body staining and non only in excision repair synthesis, as it was suggested earlier, but also may typically contained more late replicated DNA, whereas the active X chromo- be associated with other types of cellular response to UV-damage. some (Xa) contained more early replicated DNA, showing the asynchronous replication of the two X chromosomes. These results indicate that this method allows us to investigate the 3D distribution of replication sites within a chro- mosome territory. Furthermore we are investigating if the previously found difference in shape between Xi (rounder) and Xa (more extended) is main- tained during replication.

595 596 STRONGER TRANSCRIPTIONAL SILENCING IS ASSOCIATED WITH CHARACTERIZATION OF HUMAN HAT B: ACETYLATION OF MAP KINASE REGULATED SIR3P HYPERPHOSPHORYLATION. HISTONE H4 IN THE COMPLETE DEPOSITION-RELATED ((Elisa M. Stone and Lorraine Pillus.)) Department of Molecular, Cellular PATTERN and Developmental Biology. University of Colorado, Boulder CO 80309-0347 ((L. Chang*, S. Loranger*, C. Mizzen+, C.D. Allis+, and USA. email: elisalbeagle.colorado.edu pillus~spot.colorado.edu A.T. Annunziato*)) *Department of Biology, Boston College, The yeast silencing protein Sir3p plays a crucial role in the maintenance of Chestnut Hill, MA, 02167 +Department of Biology, University of telomere structure and in transcriptional repression at the silent mating-type Rochester, Rochester, NY 14627. loci and telomeres. We demonstrate that this key silencing protein is subject to dynamic phosphorylation. Our data suggest that this regulated phospho- The properties of histone acetyltransferase B (HAT B) from HeLa rylation is important for modulating Sir3p function. Sir3p phosphorylation is cells have been investigated. The enzyme is cytoplasmic, and sensitive to several critical physiological stimuli, including mating pheromone, acetylates free histone H4 but not H3. An unacetylated peptide heat shock and starvation. Genes encoding components of the pheromone representing the N-terminus of histone H4 was rapidly acetylated by response MAP kinase cascade are required for pheromone-induced Sir3p hy- the enzyme in vitro. In contrast, a synthetic peptide already perphosphorylation. Significantly, constitutive activation of the MAP kinase acetylated on the lysines at positions 5 and 12 was an extremely pathway in vivo results in simultaneous Sir3p hyperphosphorylation and sub- poor substrate, suggesting that these sites, are stantially increased transcriptional silencing at telomeres. Phosphorylation of which those acetylated in newly synthesized human H4, are recognized by Sir3p may influence subnuclear positioning of telomeres or interactions with the enzyme. This was confirmed by other silencing proteins. These observations suggest a novel role for signal microsequencing H4 that was transduction in regulation of chromatin structure and transcriptional silenc- acetylated in vitro by HeLa HAT B: lysines 5 and 12 (but not 8 and ing. 16) were radiolabeled when [3H]acetyl-CoA was included in the reaction mixture. Using monoacetylated H4 synthetic peptides, it was determined that H4 previously acetylated on Iysine-5 is as good a substrate as the unacetylated H4 N-terminus. In contrast, a peptide already acetylated on lysine-1 2 was a significantly poorer substrate. The enzyme is not inhibited by excess diacetylated H4 peptide, and has a molecular weight of -100 kDa under native conditions. (Supported by NIH grants to ATA (GM35837) and CDA (GM53512), and by a Boston College Research Grant.) Sunday. Chromatin and Chromosomes I (597-601) 103a

597 598 INSIGHTS INTO THE FUNCTION OF HISTONES H2AX AND H2AZ IN FUNCTIONAL ANALYSIS OF THE SMC-FAMILY PROTEINS IN CHROMOSOMES ((D.R. Pilch, A.H. Orr, E.P. Rogakou, V.S. Ivanova and BUDDING YEAST. ((A. V. Strunnikov.)) Unit of Chromosome Structure W.M. Bonner.)) Laboratory of Molecular Pharmacology, DBS, NCI, NIH and Function, Laboratory of Molecular Embryology, NICHD, NIH, Each of the histone families, including the H2A family, is encoded in mam- Bethesda, MD 20892-5430 malian cells by about 20 genes. While the activity of almost all these genes The SMC family is a new group of chromosomal proteins that are indispens- is linked to DNA replication, there are several histone genes whose activity is able for the formation of mitotic chromosome structure in vivo and in vitro. not. Two of these are the genes for H2AX and H2AZ, H2A species which each The importance of this group has become evident just recently, after a num- represents 5-15% of the total H2A protein in vertebrate cells. The H2AX and ber of corresponding genes have been cloned and their products characterized H2AZ proteins both differ significantly from the replication-linked H2As. The from a variety of organisms, including bacteria, fungi, invertebrate and verte- H2AZ polypeptide has 60% overall homology with other H2A species, while brate animals. Completion of the budding yeast genome sequencing project H2AX has a unique C-terminal extension. Both the H2AX and H2AZ motifs as well as the output of the ongoing programs aimed at the characterization have been conserved throughout evolution, findings that suggest that these of human and nematode genomes revealed that there are four major groups two H2A subfamilies have functions that differ from those of the replication- within the SMC-family. In budding yeast the corresponding SMC proteins are linked H2A families. Using recombinant H2A species as well as cell lines that known as Smcl, Smc2 , Smc3 and Smc4. It appears that multiple types of the produce different amounts of H2AX and H2AZ we have been able to gain SMC proteins in the same cell have at least some essential non overlapping considerable insight into the function of H2AX in chromosomes. Evidence functions. We investigated a functional significance of the major putative do- will be presented that histone H2AX is involved in monitoring the integrity of mains of the SMC proteins, including an NTP-binding region, a DA-box and chromatin using a domain strategy. two coiled-coil regions. We also assessed functional specialization of the SMC proteins through the analysis of the cytological defects associated with the corresponding smc mutations. In addition, a number of yeast proteins have been identified with a potential to interact with the SMC proteins both in vivo and in vitro.

599 600 IDENTIFICATION AND CHARACTERIZATION OF A STRUCTURE AND IMMUNOCHEMISTRY OF CHROMOSOMAL KINASE ((J. R. Swedlow and T. J. Mitchison.)) Dept. EXTRACHROMOSOMAL PERIPHERAL MATERIAL IN WHEAT CELI. S of Cellular and Molecular Pharmacology, University of California, San (((1) Yu.Chentsov, (1) V.Bourakov, (1) E.Lazareva, (2) O.Shamborant, (3) Francisco, San Francisco, CA 94143-0450 O.Zatsepina.)) (1) Department of Cytology and Histology, Moscow State We have used an in vitro chromosome assembly system to study the role University, 119899 Moscow; (2) Institute of Bioorganic Chemistry, Moscow, of phosphorylation in chromosome condensation. Interphase chromatin or mi- (3) A.N.Belozersky Institute of Physico-Chemical Biology, Moscow totic chromosomes are assembled in vitro in Xenopus egg cytoplasmic extracts We are using immunofluorescent microscopy and electron microscopy to and then isolated. Phosphorylation of interphase and mitotic chromatin pro- investigate extrachromosomal peripheral material (EPM) in wheat. By EM teins is monitored by adding -32P-ATP to either the extract or the isolated we studed the ultrastructural morphology and distribution of EPM in flat chromatin. The major phosphoproteins present in interphase chromatin iso- specimen of wheat endosperm at all stages of mitotic cycle. EPM mainly lated from 32P-labeled extracts are histones H4 and H2A.X, while the major consist of RNP-containing granules and fibers, similar with components of mitotic phosphoproteins are histones H3, B4 (a form of histone H1) and two interphase nucleoli. The layer of EPM around each chromosome is formed bands of 22 and 150 kDa. These same proteins are phosphorylated when during early prophase, then it increased in volume at the same time with ly-32P-ATP is added to isolated interphase or mitotic chromatic. Fraction- decreasing of nucleoli. In anaphase and metaphase EPM layer has the greatest ation of cytoplasm without chromatin yields no detectable kinase activity. density. EPM contain Ag-positive proteins like nucleolus. We investigate Ag- Phosphorylation of interphase and mitotic chromatin proteins is therefore cell NOR protein distribution in EPM by specific AG-NOR staining in all stages cycle specific and mediated by a chromatin-associated kinase(s). To charac- of mitosis. Besides that, some Ag-positive bands of Western-blotted proteins terize the mitotic chromosome kinase, we have added olomoucine, an inhibitor of wheat nuclei are found between 110 kD and 40 kD (110, 90, 70, 60, 55, of CDKs, and PKI and H-89, inhibitors of cAMP-dependent kinase (PKA), to 40). Nucleoli of wheat endosperm containes antigens which were recognised mitotic extracts and isolated mitotic chromosomes. Olomoucine added to mi- by autoimmune sera antibodies against components of animal nucleoli (tested totic extracts produces an interphase phosphorylation pattern on chromatin, on HeLa cells). Two of autoimmune antisera K56 (m.w. 34 kD) and pI27(m.w. but has no effect on the isolated mitotic chromosome kinase. Thus, a kinase 53 kD) were used to label wheat cells at all stages of mitotic cycle. The EPM downstream of cdc2 kinase is associated with chromosomes and responsible layer around chromosomes was stained positively in metaphase and anaphase. for chromosome protein phosphorylation. PKA inhibitors had no effect on the These results demonstrate the resemblance of proteins in plants and animals chromosomal kinase activity. We are now purifying the mitotic chromosome nucleoli and consistent with a view on EPM as an efficient way of distributing kinase. nucleolar and nuclear proteins between daughter cells.

601 A CYTOGENETIC ANALYSIS OF NUCLEAR AND CHROMOSOMAL BEHAVIOR DURING MATING AND VEGETATIVE CELL GROWTH IN A CILIATED PROTIST. ((Eric S. Cole)) Department of Biology, St. Olaf College, Northfield, MN 55057

Tetrahymena ftrmophila is a unicellular, ciliated freshwater protozoan It is well suited for undergraduate research in that cells can be grown overnight to densitIes of 100,000 cells per ml or more. These cells exhibit a wide repertoire of behaviors that can seve as model systems for investigastve analysis. Our laboratory has embarked upon a genetic analysis of the mating biology of Tetrahymena. Tetrahymens can be induced to mate in large synchronous cultures Nuclear and chromosomial cites can be monitored with DAPI stainirg and fluorescence microscopy, or In real-time with Nomarski interference microscopy. Cytoskeletal rearrangements can be followed using indirect immuno-ftuoresence and antisera to tubulin or other cytosieletal proteins. One particularly striking feature of Tetrahymena mating is that at one particular sim during conjugation, different nuclei within a common cytoplasr are simultaneously either (1) undergoing programmed nuclear elimination, (2) undergoing programmed gene rearrangement and chromosome ampitication. or (3) remaining developnnettlly inert. Another feature of Tetrahymena biology is that we can functionally distinguish sx (possibly even) different types of nuclear division based upon mutant phenotypes. Our laboratory has developed a method of screening mutations in Tetrahymena that affect genes whose products are involved (directly or indirectly) in the chromosomal and nuclear behavior associated with conjugation. Using this technology, we have isolated ten conjugation mutants affecting sarly, middle end late stages of the mating pathway. The core interest of our laboratory is to determine the developmental logic' of oilbate conjugation. That is, we hope to uncover which stages or processes are dependent on or independent of one another. Are there developmental checkpoints' goveming progreesion through each subsequent stage. and which genes control the numerous different nuclear fates within each mating partner? Our instgations have led into such diverse areas as control of chromatin condenseion, control of centromere division, apoptosis, and mcrotubula-based organele motility. Our mutest search nothonly drives our laboratory research into the control of ciliate conjugation, but it has provided a rich and broad platform to support collaborations both at our institution and in other laboratorie. We are currently involved in collab rations involving proein trafficking, programmed nuclear elimination. UV-photobiology, environmentalfy induced stress responses, and chromosomal mapping This system promises to continue providing rich research experiences for our undergraduates as well as fueling numerous cooperative efforts ooth at St Olauand at other research institutions. 104a RNA Localization (602-607). Sunday 602 603 THE DEVELOPMENT OF 'CAGED" FLUORESCENT OLIGODEOXY- ASYMMETRIC LOCALIZATION OF ASHI mRNA IN S. CEREVISIAE AS NUCLEOTIDE PROBES TO VISUALIZE NUCLEAR POLY(A) RNA DETERMINED BY FLUORESCENT IN SITU HYBRIDIZATION. TRAFFICKING IN VIVO.{(Joan C. Politz, Krishan L. Taneja, Richard A. ((Roy M. Long" 2, Ralf-Peter Jansen3, Isabel Gonzales3,Kim Nasmyth3 and Tuft Fred S. Fay, andRobert H. Singer)) UMMC, Worcester, MA. Robert H. Singer' 2)) O1 6t5 'Department of Cell Biology, University of Massachusetts Medical Center, 55 Methods for the use of oligos as hybridization tags to track mRNA Lake Av. North, Worcester, MA 01655 movement in vivo have been developed. To target poly (A) RNA in the 'Department of Anatomy and Structural Biology, Albert Einstein College of cell, an oligo dT 43mer was labelled at every tenth base with a Medicine, 1300 Morris Park Av., Bronx, NY 10461 caged", or chemically masked, fluorochrome (fl). Caged-fl oligos 'Research Institute of Molecular Pathology, A-1030, Vienna, Austria were introduced into live L6 rat cells with cationic lipid, and hybridization conditions were optimized using a modified in situ transcription technique. Caged-fl oligos internalized by cells were The yeast S. cerevisiae provides a model system to study the determination of uncaged in a 0.5 um spot in the nucleus using a UV laser. cell fate. Mother cells of S. cerevisiae are able to efficiently switch mating type Fluorescence (excited using a visible laser) was observed in cells only whereas daughter cells are not. Mating type switching is dependent on after a UV pulse, indicating that the fluorochromes remained caged (non-fluorescent) when attached to the oligo in the cellular milieu, and expression of the HO endonuclease. In daughter cells, HO expression is became sensitive to fluorescent excitation only after uncaging. To repressed by Ashlp. Ashlp is localized to the nuclei of daughter cells. We monitor the intracellular movement of the uncaged oligo, optical investigated the distribution of AshI mRNA by fluorescent in situ sections were captured at various times after uncaging using a high hybridization and found that Ashl mRNA is preferentially localized to daughter resolution, high sensitivity CCD camera. After 3D image restoration, the background fluorescence detected in the caged nucleus was cells of budding yeast. Within the daughter cell, Ashl mRNA is further subtracted on a pixel by pixel basis. This subtraction essentially localized to the distal portion of the bud. Localization of Ashlp to daughter eliminated background and allowed the location and intensity of solely cells is dependent on Shel/Myo4, She2, She3, She4 and SheS/Bnil. the uncaged fluorescence to be compared at the different times. At Shel/Myo4 is a class V minimyosin. SheS/Bnil is required for proper least two characteristic populations of uncaged oligo dT label were cytokinesis. The remaining three She genes encode novel proteins. As with tracked in the cell nucleus, a fraction that dispersed very rapidly from the point of uncaging (within 70msec) and a fraction that remained Ashlp, correct localization of Ashl mRNA is dependent on Shel/Myo4, She2, at the site of uncaging for minutes. This slower moving or stationary She3, She4 and SheS/Bni 1. Data will be presented detailing cis-acting label was sometimes present in discrete spots. These classes could determinants necessary for localization of Ashl mRNA. We hypothesize that represent a rapidly moving and a stationary population of poly A RNA correct localization of Ash mRNA is necessary for nuclear localization of and/or populations of unhybridized and hybridized oligos. Work is underway to further define these subpopulations. Ashlp in daughter cells.

604 605 REV-DEPENDENT LOCALIZATION AND EXPORT OF HIV-1 GAG/POL RNA ((M. -ACTIN MESSENGER RNA LOCALIZATION AND PROTEIN SYNTHESIS Dundr', G.H. Leno', N. Lewis2, D. Rekosh2, M.-L. HammarskjOld2 and M.O.J. Olson')) AUGMENT CELL MOTILITY ((E.H. Kislauskis, X-c. Zhu and R.H. Singer)) 'Department of Biochemistry, University of Mississippi Medical Center, Jackson MS 39216; Dept. Cell Biology, University of Massachusetts Medical School, Worcester, 2Myles H. Thaler Center for AIDS and Human Retrovirus Research, University of Virginia, MA 01655 Charlottesville VA 22908 B3-actin mRNA is highly localized in the leading lamellae of primary chicken The HIV-1 Rev protein is essential for the nuclear export of unspliced and singly spliced embryo fibroblasts (CEFs), proximal to the lamellipodia, the actin-rich region HIIV-1 mRNAs. Rev function is mediated through binding to the Rev response element of cytoplasm specialized for motility. We have proposed thatB-actin mRNA (RRE) located within the env gene. Through its nuclear export signal (NES) Rev mediates the compartmentalizes actin synthesis and thereby functions in regulating cell nuclear export ofRRE-containingHIV-I mRNAs through the cellular protein export pathway polarity and motility (Kislauskis et al., JCB 127:441, 1994). In order to which is different from the mRNA export pathway. To study the behavior of HIV-1 mRNAs, evaluate mRNA localization as a function of cell motility, the speed and gag/pol- and gag/pol-RRE-containing mRNAs were transiently expressed in stable Rev- direction of individual cells were analyzed by videomicroscopy and, after expressing CMT3 cells. Digoxigenin-labeled DNA and RNA probes complementary to a 2.2 fixation, the distribution of 8-actin mRNA in the same cells. CEFs with kb fragment of gag-pol gene were used for hybridization and detected with fluorochrome- localized B-actin mRNA moved 1.7 times faster (0.60 llm/min) than those labeled antibodies by confocal laser microscopy. The hybridization signal of gag/pol-RRE with nonlocalized mRNA (0.36 gtm/min). Antizipcode oligonucleotides mRNA in Rev-expressing cells was diffuse in the cytoplasm but colocalized with splicing directed against localization sequences in the3UTR of 6-actin mRNA, but factorSC35 and Sm U snRNPs as speckles in the nucleoplasm. In cells expressing higher not flanking sequences, delocalized the mRNA and consequently reduced the levels of gag/pol-RRE RNAs, the signal was more diffuseover the interchromatin space. speed to the same rate as cells with non-localized mRNA. Serum addition to Functional nuclear export of gag/pol-RRE mRNAs was confirmed by the presence of FHV-I quiescent cells results in rapid (2min) protrusion of lamellipodia induces both p24 in the cytoplasm. The signal for gag/pol mRNAs in the Rev-expressing cells showed a relocalization of 8-actin mRNA (Latham et al., JCB 126:1211), with a strong, punctate pattern of accumulation corresponding to individual transcripts over the subsequent increase in translocation. Antisense probed blocked this mRNA nucleoplasmic interchromatin space with no cytoplasmic signal. The nucleoplasmic localization in response to serum, and inhibited translocation, but not accumulation of gag/pol mRNAs was confirmed by localization of digoxigenin-labeled protrusion. Protein synthesis inhibitors were found to have the same poly(A) RNA. The signals for gag/pol-RRE mRNA and poly(A) RNAover the nucleoplasm quantitative effects on cell movement nd its induction by serum, as did the had similar patterns of accumulation in the absence of Rev and incells expressing atrans- antisense treatments. Therefore, it is likely that 6-actin mRNA localization dominant negative Rev mutant (ID Rev) with a non-functional NES. Although Rev and TD and protein synthesis are coupled such that translation of localized 8-actin Rev are predominantly localized in nucleoli, no signal forHIV-I mRNA was detected in mRNA augments the persistence and rate of cell motility. nucleoli. These studies indicate that there is no nuclear export of gag/pol mRNA (without RRE) even in the presence of Rev and this leads to nucleoplasmic accumulation of transcripts. Supported by NIH grants A134277 (to M.O.) and A125721 (to M.L.H.).

606 607 P-ACTIN MESSENGER RNA ANALYZED IN SITU USING HIGH INVOLVEMENT OF THE GTP BINDING PROTEINS RHO AND RAC IN RESOLUTION DIGITAL IMAGING MICROSCOPY. ((A.M. Femino, THE REGULATION OF P-ACTIN MRNA LOCALIZATION ((V. M. W.A. Carrington*, F.S. Fay*, R.H. Singer)) Department of Cell Biology Latham, Jr.,R. H. Singer, and A. F. Ross)) Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA and the *Biomedical Imaging Center, University of Massachusetts Medical 01 School, Worcester, MA 01655. 6M. P-actin mRNA localization can be induced rapidly (within 10 min) in Actin mRNA was analyzed at the single molecule level using in situ chicken embryo fibroblasts (CEFs) by serum, lysophosphatidic acid hybridization and high resolution digital imaging microscopy. ILPA), or PDGF. We have previously shown that this rapid induction through multiple signal transduction pathways (Latham et al. J. Oligonucleotide probes were each conjugated to five fluorochromes and the is Cell 126: 1211, 1994). The rapid induction of mRNA light emitted by a single probe was calibrated and used to identify Bior P-actin localization by LPA can be inhibited by C3 exoenzyme, either by in situ to single actin mRNA molecules or to quantify the hybridization addition to the media in culture or by microinjection, leading us to at on number of mRNA molecules sites of high concentration, i.e., the believe that the rho GTP binding protein was involved. This was transcribingm-actin gene. Activation of gene transcription was observed confirmed by the microinjection of the constitutively active rho after serum induction and revealed single transcripts leaving the protein, V14rhoA, which induced 1-actin mRNA localization. Rac microinjection of the transcription site. The transcripts moved through the nucleoplasm as single may also be negatively involved, since the wild-type rac 1 inhibits 3-actin mRNA localization. However molecules and then to the cytoplasm where they remain as single molecules. P13 kinase, does not appear to be involved since wortmannin an Nascentm-actin gene transcripts became visible within three minutes at the inhibitor of P13-kinase, only slightly inhibited the serum or PbGF when detected with probes targeted to the 3'-UTR. By site of transcription induction of 3-actin mRNA localization. Mutated forms of rho and rac regions of the nascent transcripts simultaneously detecting spatially distinct and unmutated ras are being microinjected to more specifically define with at least two spectrally distinct probes, a more detailed profile of the signaling proteins affecting 13-actin mRNA localization. transcription could be achieved. A typical gene at four minutes post Additionally, the induction of 3-actin mRNA localization through these P-actin signaling mechanisms involves primarily the actin cytoskeleton, since serum induction has 18 nascent transcripts in progress with five of the cytochalasin D inhibited the rapid induction of fractin mRNA transcripts completed through the end of the 3'-UTR. Using a method LPA, while nocodazole did not. This inhibition of which will resolve objects at 100 nm apart at the light microscope level, the !oca!ization by region immediately surrounding and including the transcription site could be actin filaments, at lower effective concentrations than cytochalasin examined with high resolution. The combined spatial, temporal and D. All of these data indicate that GTP binding proteins of the rho family which act on the actin cytoskeleton, also rapidly regulate quantitative information will provide heretofore unattainable information on and in transit between spatially localized gene expression in the cytoplasm in response to the events surrounding RNA metabolism within extracellular signals. intracellular compartments. Sunday. RNA Localization (608-613) 105a

608 609 SIGNALS WITHIN U3 SNORNA FOR LOCALIZATION TO XENOPUS NUCLEOLI. EVIDENCE FOR A NUCLEOLAR PHASE IN THE BIOSYNTHESIS OF THE ((T.S. Lange, M. Ezrokhi, A. BorovJagin and S.A. Gerbi)) Brown University, Division SIGNAL RECOGNITION PARTICLE. ((M.R. Jacobson, L.-G. Cao, Y.-L. Wang and T. of Biology and Medicine, Providence, RI 02912 USA. Pederson)) Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, MA 01545. What are the 'zip-codes' that target macromolecules to specific destinations in the cell? Much study has been devoted to protein traffic, and several of the The signal recognition particle (SRP) is a translational arrest machine required for protein guiding principles are now understood. However, little is known about control of secretion. SRP consists of six polypeptides complexed with an -300 nt polymerase RNA traffic. We are interested in signals for localization of RNA to the nucleolus, RNA III transcript, 7SL RNA. The intracellular site(s) of RNA the seat of ribosome biogenesis. The nucleolus contains many small nucleolar 7SL processing and SRP assembly are RNAs (snoRNAs), and several of them have been shown to be important for not known. As a first step, we have investigated the intracellular localization of fluorescent ribosomal RNA (rRNA) processing. We have done a molecular analysis of U3 7SL RNA microinjected into the mammalian cell nucleus, employing methods we have snoRNA, the most abundant of the snoRNAs, to define which sequences are devised and previously used to study the intranuclear dynamics of other RNAs (Wang etal. important for its nucleolar localization. Fluorescein labeled U3 snoRNA was 1991. Proc. Natl. Acad. Sci. US.A. 88:7391-7395; Jacobson etal. 1995. J. Cell Biol. injected into Xenopus oocyte nuclei. Fluorescence microscopy revealed that wild 131:1649-1658). Rhodamine-labeled 7SL RNA was microinjected into the nucleus of normal type Xenopus U3 snoRNA localized to the nucleoli in Xenopus oocytes, whereas rat kidney (NRK) epithelial cells. The introduced RNA rapidly localized in nucleoli (within human U3, which has the same conserved Boxes (A through D) as Xenopus U3 30 sec) after nucleus-microinjection. Control RNAs, viz. pre-tRNA and U6 RNA (also pol III but a different 'hinge' sequence (Xenopus nucleotides 61-75 which contain transcripts), did not display nucleolar localization. The nucleolar-localized 7SL RNA did not complementarity to the was somewhat less able to localize to Xenopus pre-rRNA), appear tobe confined specifically to the dense fibrillar component (DFC) of the nucleolus as oocyte nucleoli. Dictyostelium U3, which differs more markedly from Xenopus U3, revealed by parallel immunocytochemistry with a monoclonal antibody to could not localize at all to Xenopus oocyte nucleoli. Negative controls of U2 and fibrillarin, a known DFC marker. In this respect the localization of 7SL U6 snRNA (splicing snRNAs not endogenous to the nucleolus) did not localize to nucleolar RNA differs from the specific we have recently reported for Xenopus nucleoli. To define more precisely which regions are important for DFC nucleolar localization another pol III transcript, the RNA localization, mutations were made in Xenopus U3 in the highly conserved Boxes subunit of RNase MRP (Jacobson etal. 1995. J Cell Biol. 131:1649-1658). In situ and also in the non-conserved single-stranded 'hinge' region. The data from this hybridization experiments revealed the presence of 7SL RNA in both nucleoli and the study indicate that several regions are important for nucleolar localization. These cytoplasm. Our results, together with the previous finding that 7SL RNA is present in localization sequences could act as binding sites on U3 snoRNA for other isolated nucleoli (Reddy etal. 1981. J. Biol. Chem. 256:8452-8457), raise the intriguing molecules needed to transport U3 to the nucleolus, or as sites of interaction on possibility that an early phase in 7SL RNA processing and/or nascent SRP assembly occurs in U3 with other molecules already present in the nucleolus which retain U3 there. the nucleolus. The approach described here may be applied to other nucleolar RNAs to elucidate signals for their localization.

610 611 THE INTRANUCLEAR LOCALIZATION OF RNASE P RNA. (('M.R. Jacobson, mRNA IS ATTACHED TO THE CYTOSKELETON VIA MULTIPLE 'L.-G. Cao,tK. Taneja, 'R. H. Singer, 'Y.-L. Wang, and 'T. Pederson)) 'Cell Biology Group, SEQUENCE-INDEPENDENT SITES: POSSIBLE INVOLVEMENT OF A Worcester Foundation for Biomedical Research, Shrewsbury, MA 01545; andtDepartmnent of MAP lA-CONTAINING PROTEIN COMPLEX. ((C. DeFranco, M.E. Chicurel, CellBiology, University of Massachusetts Medical Center, Worcester, MA 01655. and H. Potter.)) Dept. Neurobiology, Harvard Medical School, Boston, MA Ribonuclease P (RNase P) is a ribonucleoprotein enzyme which catalyzes the 5 processing 02115 of pre-transfer RNAs. Although it has not yet been purified as an intact enzyme, mammalian RNase P consists of at least two polypeptides complexed with a340 nt RNA polymerase III A single stranded RNA-protein complexthat can form in vitro regardless transcript. The site(s) of RNase P RNA processing and ribonucleoprotein assembly are not of the RNA sequence has been identified. This complex contains at least two known. We have used fluorescent RNA cytochemistry (Wang et 1991. Proc. Acad. al. Nall. protein components: the microtubule-associated protein, MAP1A, and an as Sci. USA. 88:7391-7395; Jacobson etal. 1995. J. Cell Biol. 131:1649-1658) and in situ yet uncharacterized -170 kD species that contacts the hybridization methods to investigate the intracellular localization of RNase P RNA. RNA. A -170 kD Microinjection of rhodamine-labeled human RNase P RNA into the nucleus of rat kidney species becomes covalently attached to mRNA when proteins and mRNA are (NRK) epithelial cells or human (HeLa) cells revealed a very rapid and extensive localization UV cross-linked in vivo. V8 protease digestion of the -170 kD RNA binding in nucleoli, observed at real time in living cells. This rapid nucleolar localization of RNase P protein found in vivo and that found in vitro yields indistinguishable peptide RNA was specific to the dense fibrillar component of the nucleolus. Nucleus-microinjection fragments suggesting that these proteins are identical. Furthermore, analysis of of 5 and 3' truncated RNAs demonstrated that nucleotides 1-87 of RNase P RNA were mRNA bound to the cytoskeleton indicatesthatatachment occurs at any of a necessary and sufficient for nucleolar localization. At later times after nuclear microinjection number of sites along the length of the RNA, regardless the rhodamine-labeled wild-type RNase P RNA became predominantly distributed throughout d its translation state. the nucleoplasm, consistent with the steady-state distribution of endogenous RNase P RNA as We suggest that a complex that contains MAP IA and the -170 kD protein that determined by in situ hybridization. The in situ hybridization experiments revealed that binds RNA both in vitro and in vivo mediates the general attachment of although endogenous RNase P RNA was predominantly localized in the nucleoplasm at mRNA to the cytoskeleton. steady state, a low level could also be detected in nucleoli. The initial rapid nucleolar localization of nucleus-microinjected RNase P RNA followed by its gradual nucleoplasmic appearance suggests either a transient nucleolar phase in the maturation/ribonucleoprotein assembly of RNase P RNA or the presence of a high affinity binding site(s) for RNase P RNA in the nucleolus of mammalian cells.

612 613 RECRUITMENT OF RNA TO FOCAL ADHESION COMPLEXES. MIS LOCALIZED VIMENTIN mRNA PREVENTS CELL ((M.E. Chicurel1-2, R.H. Singer2, K. Taneja2 and D.E. Ingbert)) IDept. MOVEMENT INTO A WOUND. ((A.B. Fulton, E. Morris, Surgical Research, Children's Hospital/Harvard Medical School, Boston, and K. Evason)) Department of Biochemistry, University MA 02115 and 2Dept. of Cell Biology, University of Massachusetts Medical of Iowa, Iowa City, IA 52242. Center, Worcester, MA 01655. The mRNA for vimentin is largely even A potentially efficient and specificway of controlling gene expression is pericentrosomal, through the regulation of the localization and translation of mRNAs at, or during cell orientation to fill a gap in a cell monolayer (a near, sites of signal reception. To explore this hypothesis, we have studied wound). We had shown morphological consequences of the effects of focal adhesion formation on the subcellular distribution of this localization by targeting an epitope tagged vimentin mRNA. During focal adhesion formation, awide variety of molecules are mRNA with a actin 3' UTR, which localizes mRNA to recruited to sites of integrin binding, including proteins that are known to p regulate translation. We used 4.5 micron polystyrene beads coated with leading edges. Chicken fibroblasts expressing this specific integrin ligands to induce the formation of focal adhesion mRNA for 48 hr begin to form processes; by 72 hr many complexes in a spatially and temporally defined manner. We then used high are highly stellate with processes several cell body resolution in situ hybridizationwith fluorescently labelled probes to assess diameters in length. Control cells are unaffected. Cells whether RNAs are recruited to these focal adhesion complexes. Using an expressing misdirected vimentin mRNA fail to enter a oligo-dT probe we observed that poly-A+ RNA is recruited to focal adhesions within five minutes of integrin binding. This recruitment appears wound in the cell monolayer. The severity of the to saturate in approx. 20 min. Using probes for detecting ribosomal RNA phenotype induced by misdirected mRNA appears to we also observed a high degree of co-localization of ribosomes with poly- depend upon the presence of endogenous vimentin at with A+ RNA focal adhesions complexes. Pretreatment of cells mRNA, as SW13+ cells expressing endogenous vimentin puromycin (to disrupt mRNA-ribosome interactions) did not decrease the display more highly modified cell shapes than do SW13- recruitment of either poly-A+ or ribosomal RNAs to focal adhesions. In cells Current studies contrast, actin filament disruptors and, to a lesser extentmicrotubule address whether mistargeted disruptors, dramatically inhibited the recruitment of both RNAs. The mRNA, by producing ectopic vimentin protein, provides a recruitment of RNA to sites of signal reception may thus constitute a novel cellular address for the endogenous mRNA. mechanism for controlling localized gene expression in the cell. 106a RNA Localization (614-619). Sunday

614 615 INTERACTION OF CYTOSKELETAL COMPONENTS IN THE CHARACTERIZATION OF A 40S RIBONUCLEOPROTEIN COMPLEX LOCAUZATION AND ANCHORAGE OF HRO-TWI mRNA TO THE INVOLVED IN LOCALIZATION OF Vg1 mRNA ((Eric A. Arn, James 0. Deshler, Martin I. Highest, and Bruce J. Schnapp)) Dept of Cell Biology, ANIMAL AND VEGETAL POLES OF LEECH EMBRYOS. ((J.G. SotoI Harvard Medical School, Boston, MA 02115 (Sponsored by Eric Arn) and D.A. Weisblat2)) tDepartment of Biology, Eastern New Mexico University, Portales, NM 88130, and 2Department of Molecular and Cell VgI mRNA is localized to the vegetal cortex of Xenopus oocytes early in of California, 94720. oogenesis. A 366-nucleotide localization element (LE) in the Vgl mRNA Biology, University Berkeley 3UTR is sufficient to direct vegetal localization of RNA'injected into oocytes and is therefore to recruit all factors for the of the genus fertilization occurs competent cytoplasmic necessary In leeches Helobdella, internally but recognition, translocation, and anchoring of Vg1 RNA. In order to identify development of the zygote is arrested at meiosis I until the zygotes are these functional components of the localization machinery, we have deposited in cocoons on the ventral side of the parent leech. Rings of clear investigated ribonucleoprotein complexes formed on Vgl LE RNA in vitro. cytoplasm (teloplasm) appear at the surface of the animal pole first and Using non-denaturing gel electrophoresis we have detected a high-molecular then the vegetal pole, approximately four hours after zygote deposition. weight ribonucleoprotein complex associated with Vgl LE transcripts The teloplasm contains determinants that are necessary for teloblast incubated in "crushed" oocyte cytoplasmic extracts. The specificity of this formation and mesoderm determination. We have previously isolated a association was demonstrated by the ability of a low molar excess of Helobdella homolog of the Drosophila gene twist, Hro-twi, as the first step unlabeled Vg1 LE RNA, but not non-specific transcripts, to compete for in identifying candidate genes that may act in mesodermal determination in complex formation. Vgl LE mutants defective for in vivo localization (cf. the early leech embryo. Hro-twi transcripts are detected in the oocyte and Deshler et al, this meeting) showed altered efficiencies of competition in vitro. in the early zygote. In the present study we examined the role of In particular, a mutant (AE2) which shows no detectable localization in vivo microtubules and microfilaments in the localization of Hro-twi mRNA to displays a drastically reduced ability to compete for complex formation. The the Nocadozole and colchicine treatments of correlation between altered localization pattern and complex competition teloplasm. drug zygotes efficiency indicates a function for these complexes in mRNA localization. are suggest that microtubules involved in Hro-twi mRNA localization and The native gel-shift assay was also used to monitor purification of the Vgl LE maintenance to both poles and may protect it from degradation. Slot blot ribonucleoprotein complex from oocyte extracts. Purified complex sediments results show a significant decrease (Welch t test, P=0.0049) in detectable at approximately 40S and contains a distinct set of polypeptides identified by mRNA signal between controls and nocadozole-treated zgotes. SDS-PAGE and silver-staining. Analysis of the components of purified Cytochalasin D results suggest that microfilaments are not involved in Hro- complexes should allow the identification of individual factors involved in the twi mRNA localization, but that they may be involved in mRNA anchoring localization process. to the cortex. Finally, we show evidence suggesting that the localization of Hro-twi mRNA, but not the formation of teloplasm, is dependent on a phosphorylation event.

616 617 U3 SMALL NUCLEAR RNA LOCALIZES IN THE NUCLEOLUS VIA A COORDINATE CONTROL OF TRANSLATION AND LOCALIZATION CAP- DEPENDENT MECHANISM ((M. R. Jacobson and T. Pederson.)) OF VG1 MRNA IN XENOPUS OOCYTES ((JamesE.Wilhelm 3, Cell Biology Group, Worcester Foundation for Biomedical Research, RamanujanS.Hegde2, RonaldD.ValeW3.)) 'Departments of Pharmacology Shrewsbury, MA 01545 and Biochemistry, 2Department of Physiology, 3The Howard Hughes Medical U3 is one of several small nuclear RNAs (U3, U8, U13, etc.) defined by Institute University of California, San Francisco, CA 94143 their nucleolar localization and association with the nucleolar protein, fibril- Vgl, a member of the TGF-,3 family involved in mesoderm induction, is larin. U3 and U8 have been implicated in pre-ribosomal RNA processing. translated subsequent to the localization of its mRNA to the vegetal pole of Like the spliceosomal small nuclear RNAs Ul, U2, U4 and U5, the nucleolar Xenopus oocytes. While the localization of Vgl mRNA is known to be directed small RNAs U3, U8 and U13 carry a 2,2,7-trimethylguanosine cap at their 5' by the 3' untranslated region (UTR), the basis of its translational regulation ends. The function of this hypermethylated cap structure in mammalian small is unknown. Using a microinjection assay, we have shown that the 3'UTR of nuclear RNAs is not known. (Unlike the situation in Xenopus oocytes, the Vgl causes translational repression of two different reporter mRNAs. This trimethylguanosine cap is not required for nuclear import of Ut and U2 RNAs translational repression activity is found throughout the oocyte and at all in mammalian cells). We investigated the intranuclear traffic of U3 small nu- stages of oogenesis. Furthermore, this translational repression is not due to clear RNA by the method of fluorescent RNA cytochemistry (Wang et al. changes in RNA stability or poly(A) tail length. We have also defined a 350 1991. Proc. Natl. Acad. Sci. U.S.A. 88:7391-7395; Jacobson et al.. 1995. J. nucleotide region of the 3'UTR that is distinct from the localization element Cell Biol. 131:1649-1658). Rhodamine-labeled U3 RNAs were transcribed in and that is necessary and sufficient for translational repression. Using the 350 vitro, purified, microinjected into the nucleus of normal rat kidney cells and nucleotide translation control element (TCE) in UV-crosslinking assays, we their localization visualized by fluorescence microscopy. U3 RNAs contain- have identified a 38 kD protein that binds specifically to the TCE. We are ing a 7-methylguanosine cap rapidly accumulated in nucleoli. In contrast, no currently purifying this protein by RNA affinity chromatography in order to nucleolar localization was observed for U3 RNA carrying a 5' triphosphate. understand its role in translational repression of Vgl. Our results suggest that We conclude that nucleolar localization of U3 RNA is cap dependent. We do factors colocalized with Vgl mRNA at the vegetal pole relieve translational not know if the nucleus-microinjected, 7- methylguanosine-capped U3 RNA repression to allow expression of Vgl protein. Since Vgl is a secreted protein, becomes hypermethylated. We are currently testing U3 RNAs transcribed ei- we propose that the main role of translational repression is to promote the lo- ther with a 2,2,7-trimethylguanosine cap or other cap analogues. Our present calization of the Vgl message by preventing the signal sequence in the nascent results nonetheless demonstrate that a 5' cap structure is required for the chain from docking the message to the endoplasmic reticulum. Experiments nucleolar localization of U3 RNA. are currently underway to test this hypothesis.

618 619 LOCALIZATION OF OSKAR RNA BY NON-SELECTIVE TRANSPORT INTRANUCLEAR RING-SHAPE STRUCTURES IN LACANDONIA ((J. B. Glotzer, R. Saffrich, W. Breitweiser, E. Karsenti, M. Glotzer, and A. SCHISMATICA AND OTHER SPECIES ((Jimenez-Garcia, L.F.(1), Ephrussi.)) Programmes in Developmental Biology, Biochemical Agredano-Moreno, L.T. (1), Jimenez, J. (1), Gonzalez, M.A. (2), Instrumentation, and Cell Biology, European Molecular Biology Laboratory, Segura-Valdez, M.de L. (3), Lopez-Velazquez, G. (4), Ramos, C.H. (1), Heidelberg, Germany Martinez, E. (1).)) (1) National Autonomous Universty of Mexico, (2) Localization of oskar (osk) mRNA to the pole plasm at the posterior of National Institute of Perinatology, (3) National Institute of Respiratory the Drosophila oocyte is critical for abdomen development and germ cell for- Diseases, (4) National Institute of Pediatrics. mation in the embryo. The oocyte develops in an syncytial egg chamber, We have studied the three dimensional organization of the ring-shape struc- connected at its anterior to the nurse cells. osk mRNA is thought to be tures of Lacandonia schismatica by serial sectioning of epon embedded mate- synthesized in the nurse cells and transported into the oocyte where it ac- rial for electron microscopy. Ring-shape structures are spherical bodies with a cumulates specifically in the pole plasm, distal to the nurse cells. To study hollow filled with fibers. Thisstructure is similar to that described for rooster the mechanisms of osk mRNA transport, we have developed an in vivo assay liver stimulated with hormones. In addition, we have found ring-shape struc- that permits direct observation of injected, fluorescently labelled RNA during tures in other similar plants as 7riuris, Sciaphyla and other saprophytes as the process of localization. We show that osk RNA injected at the anterior Voyria and Gentiana. Highly evolved monocotyledons as orchids (Encyclia) of the oocyte accumulates specifically in the pole plasm. In stage 10 and 11 also have ring-shape structures. Dicotyledons as tomato (Lycopersicon) and oocytes, the fluorescent RNA moves in spiral waves that parallel the trajec- animal cell lines from marsupials (Potorous) or mouse liver cells (Mus) also tories of yolk platelets during the process of ooplasmic streaming, suggesting contain intranuclear ring-shape structures. Because several plant and animal that the streaming promotes RNA translocation. Indeed, depolymerization of cells contain ring-shape structures, we propose that they may have an impor- microtubules blocks both streaming and osk RNA dispersion away from the tant role in nuclear metabolism. injection site. However, when the RNA is injected closer to the posterior pole, depolymerization of microtubules has no effect on the localization. Moreover osk RNA injected into stage 9 oocytes is localized even in the presence of microtubule depolymerization drugs, in spite of the fact that these oocytes do not display ooplasmic streaming. We conclude that microtubules are not ab- solutely required for the localization of injected osk RNA, and that the RNA can be localized by a combination of a non-selective dispersion and binding to a receptor prelocalized at the posterior pole of the oocyte. Sunday. Translational Control (620-625) 107a 620 621 INFLUENCE OF THE 5' UNTRANSLATED REGION ON MESSENGER RNA ALTERING THE 5' UNTRANSLATED REGION OF RIBULOSE TRANSLATION ((M.M. Pedoi, M.P. Fata, A. Vbel, and E.D. iebe)) BISPHOSPHATE CARBOXYLASE SMALL SUBUNIT mRNA AFFECTS ITS DqermentofBodniay Molecular Biology, Mayo Foundation, Rochester, MN STABILITY, POLY(A) SHORTENING AND TRANSLATIONAL EFFICIENCY 55905. (Spon. by M.P. Fautac.) ((P. Liggit, J.F. Gera, and E. J. Baker)) Department of Biology, University of Nevada, Reno, NV 89557-0015. Mlmeuina pigGPIG ge codes for a polyprotein pecur that is cleaved ultimately The to yield two protein products. In the seminal vehicle, a promoter containing both a ribulose bisphosphate carboxylase small subunit-2 (RubS2) mRNA in is stable - 5 TATA and CCAAT box produces a single transcript that haa only 23 nuclootidea (nts) Chlamydomonas very (tt/2 hrs) and exhibits especially slow poly(A) shortening (< This mRNA is also of5'-usulaedrmom ('UTR). Translation of this mRNA in vitro or in vivo yields 1OA/hr). very inefficiently translated (< 30% ribosome-associated, 1-2 a singepedmi- protein product However, activation of an upstream promoter in ribosomes). The reason for its poor translation is not clear, but is the tesaprduc several novel mRNAa. Isolationof*Ye cDNA clones for novel tests known to not involve its 3' UTR sequences. Its short 5' UTR (34 nts) trata, TSMl, TSM2, and TSM3, revealed that each of them a common ha 5' appears to not carry a translational repressor element per se: teiminima as vd asueo Yr3'TRad equeceidmsc to those ofthe seminal vesice extending the 5' UTR of a well-translated a-tubulin mRNA with the trnaypt. However, the 'Rs ofthetesti transcript differ in both egth (rangig RubS2 sequence does not inhibit the translation of the resulting from 416 nts for TSM3 to 646 nta for TSMl) and sequence. Comparison of the teatia chimeric tubulin mRNA. A chimeric construct, in which the RubS2 cDNA that clatwethe smaen suggestsce themultiltests wanacript are gene is placed downstream of the inducible a-tubulin promoter, urted by altenatve splicing of an exon which codes solely for 5'UTR sequence. An produces a RubS2 mRNA with a 129-nt 5' extension. The extension unusual feature in the testia transcripts is the number of upstream AUGs (uAUGs) is composed mostly of al-tubulin 5 UTR sequences and some present before the statt of the ajor open reading frame (ORF) coding for die GPIG normally untranscribed flanking sequences. The extended RubS2 mRNA exhibits a much shortened half-life P-otei Mms ac mod f anlation initiation huggesta that mRNAs containing (30 min), accelerated kn numbe aofuAUGS ahmWdnotbetranslated However, TSMl (with 17 uAUGs), poly(A)-shortening, and more efficient translation (70% Whether these effects are TSM2 (13 uAUGs), and TSM3 (10 uAUGs) each direct the aythsis of three protein polysomal). multiple operationally related, and whether some or all are due to authentic products in rit reticulocyte These are with antiera lya. proteins precipitable "determinants" in the included tubulin mRNA sequences is under specific for he seminalvehicleproteinproducts ofGPIG. Results firm the tansltion investigation. We have found that the half-life and poly(A) ofwildtype and mutant transcripts auggeata that the three proteinproducts aise from shortening of the RubS2 mRNA are under carbon-source control, stemathve translioniitia tion at AUG triplets within the maUor ORE. Thea data lad and can be altered independently of translational efficiency. to the hypotbeaia that the 5'UTRs of TSM1-3 influence the choice of translationas ites as will as the efficiency of tranalationt

622 623 RIBOSOMAL FRAMESHIFTING IN TRICHOMONAS VAGINALlS VIRUS. TRANSLATIONAL REGULATION BY mRNA TRANSPORT ((H.W. Liu, C.H. Sun and J.H. Tai)) Division of Infectious SEQUENCE OF MYELIN BASIC PROTEIN. ((Sunjong Kwon and John Diseases, Institute of Biomedical Sciences, Academia Sinica, Carson)) Department of Biochemistry, University of Connecticut Health Taipei, Taiwan, Republic of China. Center, Farmington, CT 06032. Trichomonas vaginalis virus (TVV) is a nonsegmented double- Myelin basic protein (MBP) mRNA is transported from the cell body to the stranded (ds) RNA virus that modulates the expression of distal processes and myelin membranes of oligodendrocytes. Transport certain virulent factors in the pathogenic protozoan 7'. requires a 21 nucleotide sequence in the 3' the untranslated region (UTR) of vaginalis. Studies on genomic organization of type I TVV MBP mRNA. This sequence is referred to as the RNA transport sequence show that the translation of viral RNA-dependent RNA (RTS). A similar sequence was present in a variety of other transported polymerase (RDRP) gene in TVV begins from translation RNAs. Translation may be coordinated with mRNA transport to ensure initiation of an overlapped upstream capsid protein kCAP) gene localized protein synthesis at specific sites in the cell. To determine whether via ribosomal frameshifting. This ribosomal frameshifting was the MBP RTS can function as a cis-acting translational regulatory element, also accomplished in E. coli with much reduced efficiency, we developed an in vivo translation assay using green fluorescent protein and a small percentage of the recombinant CAP and gag-pol- (GFP) from Aequorea victoria as a reporter for translation GFP mRNA like RDRP were incorporated into virus-like particles. and chimeric GFP mRNA, containing the MBP RTS in the 3' UTR, were Deletion studies suggest that the C-terminus of the RDRP or prepared by in vitro transcription and microinjected into neuroblastoma the corresponding RNA region may be important in the B104 cells, CHO cells, and shiverer oligodendrocytes. Texas Red- morphogenesis of TVV in E. coli. conjugated dextran was coinjected to normalize for the amount of injected mRNA. Injected cells were incubated 370C for 20-24 hours and the expressed GFP/Texas Red intensities were determined using dual channel confocal microscopy. The cells which were injected with RTS containing GFP mRNA expressed approximately 10 times more GFP than the cells injected with the GFP mRNA. No significant difference in translational efficiencies for GFP mRNA and RTS-containing GFP mRNA was seen when rabbit reticulocyte lysates or wheat germ extracts were used as in vitro translational assays. These results indicate that the RTS acts as a cis-acting translational enhancer in a variety of mammalian cells.

624 625 TRANSLATIONAL CONTROL DURING THE DEVELOPMENT THE ROLE OF EUKARYOTIC INITIATION FACTOR-2 (elF-2) IN THE CONTROL ()F PROTEIN SYNTHESIS IN MURINE ERYTHROLEUKEMIA (MEL) CELLS. ((J.E. Hyde and H.A. OF VOLVOX. ((H.M. Girardin, J.A. Pelletier, L. Jovanovic, and Jenkins)) School of Biomolecular Sciences. John Moores D. Bourgaize)) Program in Cell and Molecular Biology and Liverpool University, Liverpool, U.K. Biochemistry, Colby College, Waterville ME 04901. MEL cells are erythroid precursors which can be induced to undergo programmed differentiation (Spon. by P.G. Greenwood.) similar to normal erytiroid cells. The process of differentiation is associated with a number of cellular changes including a decrease in overall protein synthesis, but with an increased expression of erythroid specific proteins, including globin. elF-2 is a key regulatory protein in the initiation of The colonial green algo Volvox exhibits a virtually complete protein translation, with phosphorylation of the a-subunit resulting in the inhibition of protein cessation of protein synthesis when entering a period of growth synthesis. It has been reported that the elF-2a kinase haem-repressed inhibitor (HRI), is induced without illumination. During this dark period, embryogenesis during differentiation of MEL cells. MEL C88 (hGH) cells have been stably transfected with a vector containing human growth hormone (hGH) cDNA and a human globin locus control region (LCR). and development require entry into the next light period, As result these cells express recombinant hOH during differentiation. However, the pattern of elF-2 effectively synchronizing the growth of the culture. We have phosphorylation in relation to MEL cell differentiation and production of recombinant proteins is not begun to explore how the shift from light to dark triggers such known. The total amount of mRNA isolated from uninduced MEL C88 (hGH) cells remained dramatic translational repression. We have determined that relatively constant during batch growth, whereas in cells induced to differentiate the total amount of mRNA decreased. However, globin and hGH mRNA increased in induced cells. The maximum rate initiation of translation is blocked in the dark, and have used a of protein synthesis in uninduced cells occurred towards the end of exponential growth. However, variety of methods to examine possible changes in the state of in cells induced to differentiate the rate of protein synthesis remained relatively constant throughout initiation factors that might correlate with changes in batch growth. Western blot of cellular protein from induced and uninduced MEL C88 (hGH) cells indicated that the total amount of eIF-2a changed during batch growth. IEF indicated that there was translational activity. Antibodies to wheat germ translation a higher proportion of the phosphorylated form of elF-2a in induced cells compared with uninduced factors have been shown to cross-react with many of the cells when there was the greatest difference in the protein synthesis rates. These data indicate that analogous factors in Volvox, and have been used to show that both changes in the total amount of eIF-2 and phosphorylation of the a-subunit may account for the phosphorylation state of several of these factors changes changes in the rate of protein synthesis in MEL C88 (hCH) cells. when the organism enters a dark period. We have also examined different signaling pathways in an attempt to elucidate the mechanism through which translational control is exerted. A model for the operation of translational control in Volvox will be presented. 108a Translational Control (626-629). Sunday

626 627 IDENTIFICATION OF TWO F-ACTIN BINDING DOMAINS ON EF-1A TRANSLATIONAL CONTROL OF CHLOROPLAST GENE THAT ARE REGULATED BY PH AND BLOCKED BY EXPRESSION ((O. Stampacchia, J.-L. Zanasco, W. Zerges, J. AMINOACYL-TRNA ((Gang Liu, Jianzhong Tang, Brian T. Edmonds, Girard-Bascou*, P. Bennoun* and J.-D. Rochaix.)) 0. Stampacchia, J.-L. John Murray and John Condeelis.)) Department of Anatomy and Structural Zanasco, W. Zerges, J. Girard-Bascou*, P. Bennoun* and J.-D. Rochaix Biology, Albert Einstein College of Medicine, Bronx, New York 10461 > We report the characterization of two nuclear PSI mutants, F15 and F24. Elongation factor 1 alpha (EF-la) is an actin binding protein and bundles The two mutants have a defect in the synthesis of the PsaA and PsaB core actin filaments with a unique bonding rule that defines a special acting fila- subunits, as shown by pulse-labeling of chloroplast proteins. Northern analysis ment compartment. This may provide a micro-environment with properties shows that the mRNAs coding for both proteins are stable in the mutants, that could be important for transport, anchorage and/or translation of mRNA suggesting a translational defect. We isolated a chloroplast suppressor of the that is also associated with actin filaments. Two domains on Dictyostelium F15 mutant: the suppressor was shown to be a single base change in the EF-la that binds to F-actin with different pH sensitivities were previously 5'UTR of the psaB gene. This mutation also destabilizes a putative secondary identified by us. By comparison of F-actin binding affinities of various EF-la structure present in the WT 5'UTR. The suppressor is unable to suppress the truncates, we have further defined the boundaries of the two F-actin binding F24 mutant phenotype, suggesting that the two nuclear functions mutated in sites in N- and C- terminal domains, respectively. When EF- la was mixed with the F15 and F24 strains have distinct roles in the translation of psaB mRNA. F-actin as EF- la:GTP:Phe-tRNA ternary complex, EF-la's F-actin bundling A chimeric gene containing a fusion of the 5'UTR of the psaB gene with the and binding activities were little affected at pH6.5 but significantly reduced aadA gene coding for spectinomycin resistance was constructed, inserted into at pH7.0, suggesting that the ability of aa- tRNA binding site overlaps with the chloroplast genome and then crossed to the mutant strains, to analyze one or both F-actin binding sites on EF-la and that the ability of aa-tRNA to its expression in the mutant nuclear context. In both crosses to F15 and block binding of EF-la to F-actin is pH regulated. We propose that increas- F24 strains, the PSI progeny was sensitive to the antibiotic, identifying the ing cytoplasmic pH, that is correlated with increases in protein translation, 5'UTR of psaB as the target for both nuclear functions. A similar chimeric reduces EF-la's F-actin binding activities thereby providing an elevated local reporter gene containing the suppressor mutation in the 5'UTR was capable concentration of EF- la supplied by the dissociating EF-la:F-actin complex. of conferring antibiotic resistance even when crossed to the F15 mutant, as This newly unmasked EF-la would be accessible to aa-tRNA and the other expected. Since the chimeric fusions contained only the promoter region and components of the translational apparatus that are associated with actin fila- the 5' UTR, we can exclude any post-translational effect: the F15 and F24 ments in vivo. mutations identify two novel nuclear functions involved in psaB translation initiation.

628 629 INTERSECTION BETWEEN THE GLYCOLYTIC AND PROTEIN DIRECT TRANSCRIPTION OF ANTISENSE AND SENSE RNA FROM SYNTHETIC PATHWAYS IN METABOLIC CONTROL: A RT-PCR cDNA PRODUCTS FLANKED BY T7- AND T-3-RNA-POLY- UNIFYING THEORY. ((Marco Rabinovitz)) NIH, Bethesda, MD 20892. MERASE PROMOTERS. ((M. Kashimata, N. Minami, and E.W. Gresik)) Dept of Pharmacol, Sch of Dent, Meikai U, Sakado, Saitama, Japan (MK, NM); Dept of Cell Biol and Anat Sci, CUNY Med Sch, New York, NY (EWG). A broad range of observations in the literature, obtained from all levels of mammalian biological organization, including the intact animal, organ A method has been devised to transcribe antisense and sense RNA from cDNAs of perfusion, cell culture, cell lysates and highly purified enzymes, can be any targeted sequence in a full cDNA library, by flanking the targeted sequence with RNA-polymerase (RNA-PMase) promoters. Such flanked cDNA products are synthe- integrated so that the pleiotypic effects of amino acid deficiency are sized from total RNA by reverse transcription (RT) and a two-step polymerase chain explained by a single mechanism. The lesions observed include inhibition reaction (PCR). A mouse brain cDNA library was synthesized from total RNA by RT of glycolysis, glucose uptake, and nucleic acid synthesis, and the with random hexamer primers. From this cDNA library, a final cDNA product of 454 bp disaggregation of cellular polyribosomes caused by a block in formation of was amplified by a 2-step PCR, encoding partial sequence of TGFa mRNA, flanked the peptide chain initiation precursor, the 43S ribosomal subunit. This by promoters for T7- and T3-RNA-PMases at its 5'- and 3-ends, respectively. The 1st subunit contains the ternary complex, whose step of the PCR required two sets of amplimers: one set corresponded to TGFa eIF-2.GTP-Met-tRNAimet, mRNA sequences only; the other set consisted of two 30-mers, each containing 23 GTP is hydrolyzed to on peptide GDP chain initiation, and requires bases of the an RNA-PMase promoter and 7 bases of the targeted TGFa mRNA replacement of the GDP with GTP for a new round of peptide synthesis to sequence. The cDNA products of this 1 st step were further amplified by the 2nd begin. The mechanism involves the inhibition of phosphofructokinase by step of the PCR, using only the 2nd set of primers including the promoter sequen- uncharged tRNA formed during amino acid deficiency and the resulting ces. Transcriptions of antisense and sense cRNAs from the promoter-flanked cDNA depletion of the enzyme's product, fructose-1,6-diphosphate. This and from linearized pmTGFa373 (a recombinant pBluescript KS/+ plasmid with a 373 bp insert for part of TGFa mRNA) were compared. Transcription by T7-RNA-PMase diphosphate is an precursor for the synthesis of early from the cDNA or the RE-cut plasmid resulted in 48.3% and 46.4% incorporation of phosphoribosylpyrophosphate (PRPP) and cofactor of eIF-2B, the 32P-UTP, with yields of 6.24 sg and 6.00 pg antisense cRNA, respectively. With T3- guanine nucleotide exchange factor. PRPP is required for synthesis of RNA-PMase-driven transcription, incorporation was 37.2% and 40.8% with the cDNA nucleotides for RNA synthesis and eIF-2B is required for the exchange of and the linearized plasmid, respectively, with corresponding yields of 4.86 pg and GDP for GTP indicated above. A diagram of the mechanism will be 5.27 pg sense cRNA. Thus, this method allows synthesis of cDNA flanked by two RNA-PMase promoters that are in efficiency to linearized plasmids presented and the experimental data and literature in support equal for of each synthesis of cRNAs. (Support by NIH Grant DE10858 to EWG). step will be given. Cell Interactions in Development (630-631).

630 631 INTERACTION OF THE CELL INTRINSIC DETERMINANT NUMB ROLE OF LATERAL AND VENTRAL MESO-ENDODERM IN AND THE NOTCH RECEPTOR FOR CELL-CELL COMMUNICATION ANTEROPOSTERIOR PATTERNING OF THE ZEBRAFISH NEURAL AXIS. DURING ASYMMETRIC CELL DIVISION ((Ming Guo, Lily Yeh Jan and ((Katherine Woo, Scott E. Fraser)) Division of Biology and Beckman Institute, Yuh Nung Jan,.)) Howard Hughes Medical Institute, Departments of Caltech, 139-74, Pasadena, CA 91125 Physiology and Biochemistry, University of California at San Francisco Both Numb, an inherited determinant, as well as a transmembrane receptor Previously, we fate mapped the presumptive neurectoderm of the zebrafish and Notch and its ligand Delta which mediate cell-cell communication, are uti- found regionalization of neural progenitors in the shield-stage gastrula(6 hr lized in cell fate specification during asymmetric divisions in the Drosophila postfertilization; Woo and Fraser, Development 112: 2595-2609): presumptive peripheral nervous system. During this process, a sensory organ precursor forebrain progenitors are found mainly in the medial portion of the dorsal cell undergoes rounds of asymmetric divisions to generate four distinct cells ectoderm, and presumptive hindbrain progenitors are located more laterally. of a sensory organ. In order to understand how cell intrinsic determinant Here, we further explore this early patten by identifying the tissues that may be interfaces the machinery for cell-cell communication during asymmetric divi- responsible for this early regionalization. We find specific effects of the lateral and sion, we investigate the interactions among Notch, numb and tramtrack (ttk) ventral meso-endoderm on the patterning of the neural axis in stages as early as which encodes a transcription factor. Firstly, we show that Notch plays a crit- 6 hr postfertilization. When presumptive forebrain progenitors are transplanted to ical role during asymmetric divisions in embryogenesis. Secondly, numb and close to the lateral meso-endoderm, they participated in the formation of the Notch most likely act in the same genetic pathway in which numb negatively hindbrain. In the converse experiment, when lateral meso-endoderm tissue is regulates Notch. Direct protein-protein interaction between Numb and Notch transplanted to presumptive forebrain region in the gastrula, it transforms the is detected by both yeast two-hybrid assay and in vitro binding. Two regions region into hindbrain-like structures, as evident by the ectopic induction of otic of Notch and the phosphotyrosine binding domain of Numb are required for placode, and ectopic expression of the homeobox gene Krox2O, a marker for this physical interaction. Thirdly, Notch positively regulates ttk. This leads rhombomere 3 and 5, in the forebrain. Surprisingly, we also find ventral meso- to Ttk expression in the daughter cell which does not inherit Numb. Thus the endoderm to process this transforming ability. Previous experiments with inherited determinant Numb exerts a bias in the machinery for cell-cell com- amphibian embryos support a model that the patterning of the neural axis results munication to allow the specification of distinct daughter cell fates. Ttk acts from two interacting signals from the axial mesoderm: an 'activator signal that neuralizes and induces anterior neural as a readout to integrate signals derived both from a cell intrinsic determinant tissue (forebrain and midbrain), and a and from cell-cell communication. 'transformer signal that converts some of the activated tissue into more posterior types (hindbrain, spinal cord) (cf. Donaich, Cell 83: 1067-1070). Our results indicate that in the zebrafish, the 'transformer' signal is not confined only to the dorsal meso-endoderm tissue. We are currently further characterizing these surgically-manipulated embryos to gain insights into the nature of these neural patterning events. Sunday. Cell Interactions in Development (632-637) 109a

632 633

EXPRESSION OF LI-CADHERIN DURING EMBRYONIC GROWTH AND NORPHOGENESIS OF OVARIAN NESOTHELIAL CELLS IN DEVELOPMENT OF THE INTESTINAL EPITHELIUM. ((B. Angres, INTERACTIVE CULTURE WITH GRANU.OSA, LUTEAL A1ID STRONAL CELLS. B. Kosel, L. Kim, A.-M. Lajous-Petter, N. Schnoy, R. Gessner and R. ((S.V. Nicosia, B5O. Saundorsand L.B. Mora )) Departments of Tauber)) Institut fur Klinische Chemie und Biochemie, Virchow-Klinikum Pathology, Univ. of South Florida and Moffitt Cancer Certer, Tempa, FL 336121 and Univ. of Los Andes, Merida, Venezuela'. der Humboldt-Universitkt zu Berlin, 13353 Berlin. At variance with adjacent mosothelia, the ovarian mesothelium Adhesion mediated by different members of the cadherin family plays is throughout life a morphogenetically active tissue that gives morphoregulatory roles during development of many tissues. We recently rise to coaon epithelial tumors including papillary serous neoplasms. To gain further insight into the regulation of this identified a novel member of this protein family, termed LI-cadherin, that is tissue, ovarian mosothelial (OKC) and submesothelial stromal expressed in liver and intestine in rat (Berndorff et al., 1994, J. Cell Biol. (SC) cells were isolated from NEW rabbits by stepwise dispersion 125:1353-1369). When compared with classical cadherins its extracellular (In Vitro Coll Dev Bial 31:300, 1995); granulosa cells (CC) were domain exhibits two additional cadherin repeat domains while the aspirated from mature follicles and luteal cells (LC) were harvested with collagenase from 5-day-old corpora lutea. cytoplasmic domain is very short (18 amino acids) and does not bind After a week-long in vitro expansion, ONC were cultured alone and with catenins. This suggests that LI-cadherin mediates cell adhesion independent SC, OC or LC, together in multiwell plates (+) or on filters from the actin cytoskeleton and therefore may hold a different within bicameral chambers (/), in serumless fibronectin-rich HL- morphoregulatory potential. As a first step of analyzing the role of LI- 1 medium, and were exposed to 10-5 M bromodeoxyuridine (BrdU) to cadherin during embryogenesis we studied the expression pattern of LI- study proliferation. After 7 days, all ONC expressed low cadherin during mouse development As revealed by Northern blot and molecular weight cytokeratin and focally aggregated to form papillary-like processes (PP). ONC, alone or immunohistochemical analysis LI-cadherin expression begins at day 12.5 of filter-bound, displayed a biphasic, epithelioid and spindle, cytology while gestation. The expression is confined to the intestinal epithelium at all stages *pithelioid ONC nests surrounded by negative or weakly positive up to adulthood. Electron microscopic analysis shows that LI-cadherin is SC, CC or LC were observed in direct contact cultures. BrdU localized in polarized cells at lateral cell-cell contacts but is excluded from imunoperoxidase revealed mean proliferation indices of 14.58%, the adherens junctions where to 15%, 14.97% and 21.26% (P<0.0001) in OIC alone, ONC+GC, OXC+LC E-cadherin is known be concentrated. and ONC+8C, and 27.99%, 10.38%, 10.93% and 49.15% (P<0.0001) in During days 14 to 19 of gestation the mouse intestinal epithelium is OKC alone, ONC/GC, OMC/LC and OXC/SC, respectively. The number transformed from an undifferentiated stratified epithelium into a single of PP was also significantly higher in ONC-SC and ONC/SC (P< layered columnar epithelium. Thus LI-cadherin is expressed at a very critical 0.001) than in other co-cultures or ONC alone. These data stage of intestinal epithelial development when cells begin to polarize and support the existence of stimulatory and inhibitory intraovarian paracrine signals for ONC growth and morphogenesis. differentiate while rearranging into a new morphological pattern. Repetitive activation of these signals, such as with incessant ovulation or ovarian infertility, may play a role in ONC pathobiology.

634 635 GENETIC CONTROL OF GASTRULATION INITIATION IN C. ELEGANS. ((Jennifer Knight UNC-62 IS LIKELY TO BE A REGULATOR OF MUSCLE AND HYPODERMAL and William B. Wood)) MCD Biology, University of Colorado, Boulder, CO 80309-0347. ORGANIZATION IN C. ELEGANS. ((D.C. Weaver, L. Edgar, and W.B. Wood)) MCD Biology, University of Colorado - Boulder, Boulder, CO 80309-0347 The genetic control of gastrulation is not well understood in C. elegans. It is normally initiated by the migration of the two gut precursor cells (E blastomeres) into the embryo at the Several mutant alleles of the unc-62 gene result in severe disruption of posterior patterning of 26-cell stage. We have isolated a temperature-sensitive mutation, ct226fs, which results in the C. elegans embryo (Nob phenotype). To characterize the primary defect of these animals, failure to initiate gastrulation. The mutation, tentatively defining a gene which we propose to we used the anti-LIN-26 antibody (which stains all hypodermal nuclei) and the MH27 antibody call gad-1 (gastrulation defective), exhibits a strict maternal effect. At 25°C, 100% of self- (which outlines tight junctions between cells) to examine the distribution and number of progeny embryos laid by homozygous hermaphrodites arrest with a gastrulation defect: the E hypodermal cells at various stages of embryogenesis, particularly at the start of blastomeres divide early, do not migrate into the embryo, and the embryo does not undergo morphogenesis. Self-progeny embryos from mothers homozygous for either unc-62(ct344) or gastrulation. Embryos arrest without obvious morphogenesis, but have the correct number unc-62(f2012) have -30 fewer hypodermal cells than wild type and mislocalize the P, V, and C- of cells and have fully differentiated but abnormally positioned gut, muscle and hypodermis. derived hypodermal cells (the cells that sheath the majority of the worm's body). We have mapped ct226 to the center of LG V. This region is uncovered by the deficiency Immunofluorescence staining of body wall muscle myosin shows that these embryos are also nDfI8, and cf2261nDf18 hermaphrodites lay substantially more inviable eggs at 160 than do unable to assemble the body wall muscle quadrants correctly. A 15 kb region of genomic DNA ct226 homozygotes, suggesting that cf226 is a hypomorphic allele. We have narrowed the on chromosome V contains sequences sufficient for extragenic complementation of mutations location of ct226 on the physical map to a small region between the left endpoint of nDfl8 in unc-62. Five transcripts are predicted in this region: one appears to be a novel transposon and dpy-11, and are currently injecting appropriate cosmids in an attempt to rescue ct226 and the other four comprise two related pairs of nearly identical genes. At least one member of homozygous mutant animals. Previously identified (but not well characterized) maternal- each gene pair is transcribed based on the presence of the corresponding cDNA in an effect gastrulation-defective mutants have a similar, but overall less severe phenotype than embryonic library. All of these genes display significant similarity to a human protein (pl9sk'n) ct226. We have also found that when embryonic transcription is blocked by injection of ama- which directly regulates the activity of CycA-CDK2 kinase and thereby controls entry into S 1 antisense RNA (Powell-Coffman, Knight and Wood, submitted), embryos arrest with a phase (ref. 1). RNA blot analysis shows that these genes are highly expressed in early similar phenotype to that of ct226, indicating that embryonically transcribed genes must also embryogenesis and are expressed in adults (though not in the germ line). By studying unc- be required for gastrulation initiation. Our immediate goal is to clone and molecularly 62's role in morphogenesis, we hope to understand a potentially novel mechanism of characterize gad-1. We also intend to search for embryonically transcribed genes required developmental regulation. for gastrulation in the hopes of understanding the functional relationship between maternally 1. Zhang, H. et al. (1995) Cell 82:915-925. and embryonically expressed genes in control of this process.

636 637 FORMATION OF THE ECTODERM-ENDODERM BOUNDARY A NOVEL SHIFT IN SUBCELLULAR DISTRIBUTION OF NOTCH DEFINES THE IN THE SEA URCHIN EMBRYO ((C. Y. Logan and D. R. PRESUMPTIVE ENDODERM-MESODERM BOUNDARY IN THE SEA URCHIN Duke Durham NC. 27708 EMBRYO ((David R. Sherwood and David R. McClay)) DCMB Group, Zoology McClay)) Zoology Department, University, Department, Duke University, Durham, N.C. 27708

We have shown previously through a variety of microsurgical We are interested in the molecular basis of cell-cell interactions involved in the perturbations that the archenteron is highly regulative and that generation of cell types in the sea urchin embryo. We have identified a homolog of patterning of the endoderm is accompanied by active cell signaling the Notch receptor in the sea urchin Lytechinus variegatus, and generated (McClay and Logan, Development 122:607-616, 1996). For example, polyclonal antibodies to multiple regions of this Notch. Western blot and whole- archenteron cells are uncommitted throughout gastrulation and mount localization using confocal microscopy reveal that Notch is expressed at low removal of the archenteron results in complete replacement by the levels during cleavage-stages and the early blastula stage, and that the protein is remaining circumblastoporal tissue. There appears to be a limit to gut localized to the basal and lateral surfaces of cells in the early embryo. During the forming competence, however, since removals of large amounts of mesenchyme blastula stage, however, Notch expression increases dramatically, vegetal tissue result in truncated gut structures. In this study, we and there is a shift in localization to high levels of apically distributed Notch in a ring of cells in the Cell counts were made of cells examine how the limits of the endoderm are maintained during vegetal plate. expressing apically that the of the localized Notch and cells lacking this expression pattern and compared to a recently gastrulation. We first tested the hypothesis separation published fate map of the mesenchyme blastula-stage vegetal plate (Ruffins and ectoderm and endoderm is accomplished prior to gastrulation and that Ettensohn, Dev 122: 253-263, 1996). We find that high levels of apically localized subsequently each tissue is committed along its respective pathway of Notch are confined to the endodermal precursor cells, while expression Is absent in differentiation. As a test, we transplanted ectodermal cells into the the mesodermal precursor cells which lie at the center of the vegetal plate. Lithium endoderm at the early gastrula stage. We find that ectodermal cells chloride (LiCI) is known to expand the endoderm and increase the abundance of can make endoderm when moved into the archenteron, suggesting that some mesodermal cell-types. Consistent with the expression of apically localized ectodermal cells are not committed at the time of transplantation. Notch at the endoderm-mesoderm boundary in the unperturbed embryo, LiCI However, cells at the ectoderm-endoderm junction form ectopic tissues treatment causes an increase in the number of cells that lack Notch expression in when placed within the endoderm. These data suggest that there exists the center of the vegetal plate, and an expansion of cells that express Notch apically in a ring. This apical localization of Notch in embryonic epidermal cells is novel; in a distinct boundary at the border between the ectoderm and endoderm Notch is confined in cells to areas of cell-cell may the two tissues This Drosophila, embryonic epidermal that keep separated during development. contact J. Cell. Biol. 113: This localization in a of cells whose (Fehon, et. al., 657-669, 1991). apical boundary appears to consist of population 1) the sea urchin is also because all identifed for Notch to date are in the rest of the ectoderm and intriguing ligands properties are different from the cells 2) transmembrane proteins, and it is unclear how a transmembrane ligand on a who may be unresponsive to the planar signaling that occurs within the neighboring cell could interact with a cell expressing apically localized Notch protein. endoderm. lla a Cell Interactions in Development (638-643). Sunday

638 639 MORPHOGENETIC MECHANISMS OF EPITHELIAL INVAGINATION: PRIMARY CELL-CELL FUSION IN THE SEA URCHIN PRIMARY MESENCHYME INVAGINATION IN THE SEA URCHIN EMBRYO. ((E.M. Laxson and J.D. Hardin)) Program in ((P. G. Hodor, C. A. Ettensohn.)) Department of Biological Sciences, Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706 Carnegie Mellon University, Pittsburgh, PA 15213 Little is known about cell-cell fusion mechanisms, although they are involved Invagination of epithelial tissue occurs during gastrulation, neurulation, and in such basic developmental processes as fertilization and myogenesis. In our organogenesis in many organisms. However, the underlying morphogenetic model system, the sea urchin embryo, primary mesenchyme cells (PMCs) form mechanisms of invagination are not understood. To elucidate these mechanisms, we a syncytium within which the larval skeleton is deposited. Taking advantage are the initial of the plate in the sea urchin analyzing invagination vegetal embryo, of the unique features of this system, we have developed a method for directly a process termed primary (1°) We have examined the patten and invagination. observing the dynamics of PMC fusion in vino. Fluorescein-dextran was mi- function of the distinct cell shapes found at the vegetal plate as well as the role of croinjected into single PMC progenitors (the micromeres) and embryos were the extracellular matrix (ECM) in this process. At the onset of invagination, a ring followed by time-lapse confocal microscopy during gastrulation. Cell fusion of cells with highly constricted apices (bottle cells) encircles a group of 4-8 round, was monitored by transfer of the dye from labeled to unlabeled PMCs. Fusion center cells. Surrounding the ring of bottle cells are cells highly stretched in their was first detected about 2 hours after PMCs ingress and start to migrate within radial axis. To investigate the morphogenetic role of each of these three cell the blastocoel. Fusion in with the of the populations in the process of 1° invagination, we have undertaken a series of laser proceeds parallel assembly PMC ring and is the a ablation studies in which different proportions and patterns of each cell type are pattern, complete by early gastrula stage. The existence of single, ablated and the effects are recorded using 4D microscopy. Our results reveal that complete syncytium was confirmed by Dil diffusion within the plasma mem- brane in fixed When were elimination of a 900-1800 arc of the bottle cells retards invagination of that embryos. single micromeres isolated and cultured portion of the vegetal plate but does not affect the process in the unperturbed in unsupplemented sea water, they divided and their progeny underwent fu- region. Ablation of the round, center cells or a 900-1200 arc of the stretched cells sion. This shows that the capacity to fuse is autonomously programmed in does not affect 1° invagination. Finally, a radial ablation pattern extending from the micromere-PMC lineage early in development. PMC transplantations at the center of the vegetal plate to its periphery does not affect the process. These late embryonic stages revealed that these cells remain fusion-competent long studies indicate that the number and arrangement of the bottle cells are the most after normal fusion is completed. At these later stages a group of secondary critical factors for proper invagination to occur. Further ablation experiments are mesenchyme cells, the blastocoelar cells, are also present within the blastocoel, currently underway. In addition, we have found that the ECM plays a crucial role in and are migrating and fusing with one another. This implies the existence of a Invagination. Initial perturbational studies of the apical ECM using blocking mechanism to provide the clear fusion specificity shown by the two cell types. antibodies against the protein hyalin, a major constituent of the apical ECM, suggest that bottle cell formation is prevented and invagination does not occur in antibody treated embryos. Experiments involving perturbation of the basal lamina using antibodies raised against the sea urchin laminin protein are underway.

640 641 THE DEVELOPMENT OF THE HIS-PURKINJE SYSTEM: INHIBITION OF LENS-FORMING POTENTIAL IN NON-LENS HEAD ECTODERM ELECTROPHYSIOLOGICAL AND ANATOMICAL CORRELATIONS IN FROM CHICKEN EMBRYOS. ((C.H. Sullivan, J.D. Brown, and L.J. Miller)) THE EMBRYONIC CHICK HEART ((E. Thomas Chuck, David S. Department of Biology, Grinnell College, Grinnell, IA 50112 Rosenbaum, Michiko Watanabe.)) Case Western Reserve University, Ohio During the process of lens induction in chicken embryos, head ectoderm acquires Cleveland, lens-forming potential that can be detected when pieces of ectoderm are grown in In the mature heart, the His-Purkinje system (HPS) plays an important vitro. Explants of presumptive lens ectoderm (pie) readily differentiate into lentoids role in the propagation of cardiac action potentials from the atria to the ven- and express high levels of the major lens protein, delta-crystallin (cr). We used tricular myocardium but is not present during early cardiac morphogenesis. immunoblot analysis to measure accumulation of delta-cr in a culture and compared We have previously shown using electrophysiological techniques that the HPS levels with known amounts of delta-cr standards. Almost 100% of the explants of begins to function in the chick embryo around embryonic days (ED) 6 and 7. stage 10 pie synthesized delta-cr after five days, with an average of 340 ng delta-cr These developmental times coincide with the separation of myocardium at the per culture. Although head ectoderm (he) posterior to the optic vesicles and anterior atrioventricular junction (AVJ) and the histological identification of the HPS. to the heart does not synthesize delta-cr in vivo, explants of he also synthesized delta-cr in of To determine how closely the appearance of HPS-like conduction is related nearly 100% the cultures. However, the response of pie and he cultures was different in three after 5 delta-cr accumu- to these anatomical changes, histological sections were studied using mono- important ways. First, days, lation in he reached an average of only 70 ng. Second, the time course of delta-cr clonal antibodies to titin to identify myocardial cells; monoclonal antibodies accumulation was slower in he than pie. After two days of culturing, 90% of pie to HNK-1 and the polysialylated form of the neural cell adhesion molecule explants were positive with an average of 21 ng delta-cr while 90% of the he explants (PSA) were used to identify the HPS. RESULTS: The AVJ myocardium re- were negative or only very weakly positive. Third, head ectoderm eventually iost its mained continuous at ED 5 but thinned from ED 5 until ED 8, when the ability to express delta-cr in culture by stage 13, but pie continued to synthesize high myocardium became disconnected. In the ventricles from ED 5 to 8, PSA levels of delta-cr both in vivo and in vitro. Previously we demonstrated a role for the was present in the ventricular trabeculae and in two areas in the myocardium head mesenchyme in inhibiting the lens-forming potential of he and we are testing that resembled Purkinje fibers and bundle branches respectively. HNK-1 ex- the hypothesis that neural crest cells might be the source of this inhibitory signal. pression in the ventricular conduction system was negative until ED 7.5 when We used whole-mount immunohistochemistry with HNK-1 antibodies to study the distribution of it was upregulated next to the junction of the PSA-positive bundle branches neural crest cells. Neural crest cells are found under a small region of lateral head ectoderm at stage 10. Between stages 11-13, the extent of migration of in a pattern that resembled a common bundle. CONCLUSIONS: Changes in neural crest cells continues towards the ventral midline, and by stage 13, neural crest myocardial continuity of the AVJ and in the expression of HNK-1 and PSA in cells from the left and right sides of the embryo have almost met. There appears to the ventricular conduction system occur around the same time period when be a good correlation between the infiltration of neural crest cells under head HPS-like conduction is first observed. These data suggest a complex, coordi- ectoderm and loss of lens-forming potential in this tissue. Interestingly, the close nated process governs the morphological and functional development of the association of the optic vesicle and pie appears to block migration of neural crest cardiac conduction system. cells under the pie, perhaps explaining why lens-potential is not inhibited in the pie.

642 643 STRUCTURAL CHARACTERIZATION OF THE PERIPHERAL NEURAL RETINA FURTHER EXAMINATION OF PERTUSSIS TOXIN INHIBITION OF PROTEIN, RETINA COGNIN REVEALS HIGH STRUCTURAL AND EPITHELIAL-MESENCHYMAL TRANSFORMATION IN THE EMBRYONIC FUNCTIONAL HOMOLOGY TO PROTEIN DISULFIDE ISOMERASE (H.P. CHICK HEART. ((A.S. Boyer, C.P. Erickson and R.B. Runyan)) Department Parlser, R.E. Hausman. Department of Biology, Boston University, of Cell Biology & Anatomy, University of Arizona, Tucson, AZ 85724. Boston, Ma.) During early development, progenitors of heart valve are formed by Retina cognin, (R-cognin (5OkDa)) is a transiently expressed protein localized epithelial-mesenchymal transformation of endothelial cells in the in developing chick retina and has been shown to mediate cell-cell recognition atrioventricular (AV) canal and outflow tract areas. Previous studies showed that are essential for and neuronal differentiation. The partial cDNA clone for R-cognin is homologous TGFSs this transformation. An earlier study also indicated that pertussis toxin inhibits transformation to chicken protein disultide isomerase (C-PDi (57kDa)), at least from the 3' end epithelial-mesenchymal up (Cell Regulation 1:301-313, 1990). This examines in detail the effect of to near the boundary between exons 1 and 2. As with PDi, has study R-cognin protein pertussis toxin on AV canal epithelial-mesenchymal transformation. Pertussis thioreductive activity. Here we show the homology of the two proteins through toxin blocks receptor-mediated signal transduction by ADP-ribosylating the a peptide digest fingerprinting, amino acid sequencing, and thioreductive activity subunit of Gi proteins, therefore uncoupling receptor-G-protein interaction. At assays. Although the fingerprints of C-PDI and R-cognin are quite similar, there all stages of transformation, stage 14, 16, and 18, transformation was almost are some apparent differences. The PDi fingerprint has a number of high completely inhibited. There were no mesenchymal cells at stage 14, and very molecular weight peptides not seen in R-cognin which may account for the 7kDa few mesenchymal cells at stages 16 and 18. Endothelial cell-cell separation mass difference between the two proteins. When probed with a monoclonal was inhibited at all three stages by pertussis toxin. The expression of a matrix antibody recognizing a C-terminal, endoplasmic reticulum localization peptide, the protein marker, JB3-antigen (fibrillin), and the cell adhesion molecule integrin C-PDI digest reveals a peptide containing this sequence while the KDEL sequence a-6 were both greatly inhibited by pertussis toxin in endothelial and is lacking in R-cognin. This could account for the cell surface localization of R- mesenchymal cells. The expression of transcription factor mox-1 was cognin contrasted with the endoplasmic reticulum localization of C-PDI. Finally, we inhibited in mesenchymal cells, but not in endothelial cells. The pattern of are determining whether R-cognin has one or two thioreductive sites through markers in cultures perturbed by pertussis toxin differs from cultures treated with TGF11 thioreductive activity assays and radiolabling the active sites with iodoacetate. Type IIreceptor antibodies. The conclusions from this study are: 1. Pertussis toxin blocks cell activation, cell transformation, and cell migration; 2. Since pertussis toxin can block endothelium activation after "activation" is supposed to be initiated, signal transduction by Gi may be required continuously, or at one or more critical steps during "activation"; 3. Pertussis toxin sensitive and TGF13signal transduction pathways appear to be diverged in regulation of various phenotypic markers among endothelia and mesenchymes; 4. Activation is not a single step process in response to myocardial stimulation. Sunday. Cell Interactions in Development (644-649) lila

644 645 CELL MOTILITY UNDERLYING NEURAL CONVERGENCE AND EXTENSION IN DISCOVERY OF A NEW FAMILY OF C-TYPE VERTEBRATE LECTINS XENOPUS LAEVIS ((T.M. ELUL13, M.A.R KOEHL1,2,AND R.E.KELLER3)) Jin-Kyu Lee, Kelley Moremen, and Michael Pierce Dept. of Biochemistry and 1Biophysics Grad. Group, UC Berkeley, 2Integrative Biology, UC Berkeley, Berkeley CA Molecular Biology and Complex Carbohydrate Research Center, Univ. of 94720, 3Biology Dept., University of Virginia, Charlottesville VA 22903w Georgia, Athens, GA 30605

Convergence and extension is a morphogenetic process occurring during embryonic The Xenopus laevis oocyte lectin (XL35) is packaged in cortical granules and development of many organisms in which a tissue narrows along one axis while elongating is released at fertilization to cross-link glycoproteins in the egg jelly and form a along a perpendicular axis. In the Xenopus laevis late gastrula embryo the neural ectoderm and layer of the fertilization membrane l1,2]. The lectin is found as a multimer of the mesoderm tissue regions undergo convergence and extension. Cells in these tissues -500 kDa in solution and requrires Ca + for binding activity. We have cloned rearrange or medio-laterally intercalate during convergence and extension such that the number this soluble lectin and shown that it consists of a polypeptide of 35 kDa, anc of cells along the medio-lateral axis decreases while that along the antero-posterior axis its heterogeneity on SDS-gels is due to variation of N-linked oligosaccharide increases. In order to characterize the cellular mechanism ofconvergence and extension we expression. The activity of the lectin is assayed by trypsinized erythrocyte have developed two types of explants that undergo convergence and extension when isolated agglutination, and the binding is calcium dependent and most likely to an from an intact embryo (Elul et. al., 1995). One type ofexplant consists of a single layer of epitope with terminal ac-galactose. By Northern blot analysis, mRNA for the neural deep cells (type I) while the other consists of a layer of neural deep cells overlying lectin undergoes a burst of expression at gastrulation, and early tadpole mesoderm tissue(type II). (Neural epithelial cells have been removed.) Type I explants have a stages show tissue-specific expression, assayed by in situi hybridization slower rate of extension than do type II explants. We have investigated the cell motility Two homologs of XL35 have been detected ih human tissues, and a Northern underlying medio-lateral rearrangement in these explants, using time-lapse video-recording of blot analysis demonstrates selective expression of one homolog (HL-3) in fluorescently labeled neural deep cells. We have developed a new morphometric method to heart, small intestine, colon, thymus, with lower levels in ovary, testis, ana quantify protrusive activity of cells in video-recordings. Our analyses reveal that neural deep spleen. A few other tissues show low levels of expression: skeletal muscle cells exhibit medio-laterally oriented protrusive activity and are medio-laterally elongated placenta, and spleen. The other homolog (HL-13) is expressed only in smali during convergence and extension. Cells in type I explants appear to have more protrusions/hr intestine. The human homologs are the same size as XL35 and show 60/, than do those in type II explants. We are currently investigating whether the onset ofmedio- identity at the amino acid level. Bacterial expression of both recombinant XL35 laterally oriented protrusive activity is spatio-temporally regulated in these explants. and recombinant human lectins yield proteins which, after purification ana Our results demonstrate that neural deep cells are sufficient to drive convergence and renaturation, actively agglutinate trypsinized erythrocytes. The agglutination is extension. The rate of extension and cell motility differs in neural deep cell explants with specifically inhibitnby EDTA and melibiose, hallmarks of native XL35 activity mesoderm underneath and in those without. The mesoderm may mechanically and or None of the lectins contain the common C[calcium-dependent]-type lectin molecularly induce a change in cytoskeletal or cell-adhesion molecules involved in neural deep domain. Studies are in progress to determine which cells express the lectins cell motility. Moreover, mesoderm deep cells also exhibit medio-laterally oriented protrusive and the locations of the secreted lectins, to characterize their oligosaccharide activity during convergence and extension but they are more elongated than the neural deep ligands, and to test the hypothesis that the lectins function in intercellular cells. (Supported by NIH Grant HD25595 to R.E.K and an HHMI Predoctoral Fellowship to adhesion during morphogenesis and in the adult. 1 Roberson & Barondes, 1982 JBC T.M.E) *address for correspondence 257, 22:7520; 2 Nishihara et al., 1986 Biochem. 25:6013.

646 647 CHARACTERIZATION OF SIALOMUCIN COMPLEX IN THE RAT EXPRESSION OF A JAGGED CDNA HOMOLOGUE IN THE UTERUS DURING EARLY PREGNANCY. ((R.R. McNeer and K. EMBRYONIC MOUSE SUBMANDIBULAR GLAND ((R.I. Couwenhoven Carraway.)) Department of Cell Biology, University of Miami School of and Jia Di Hu.)) Univ. of Maryland at Baltimore/ Dept of Oral Pathology Medicine, Miami, FL 33101 Mesenchymal-epithelial tissue interactions govern tissue morphogenesis and Blastocyst implantation in the uterus occurs if and only if the uterus is cell differentiation programs of several developing mammalian organs includ- in a receptive state, a phenomenon which is regulated by ovarian hormones. ing the salivary glands. Experimental evidence suggested that an EGF-like During this time, the apical surfaces of the endometrium which are normally ligand participates in the transduction of inductive signals between embryonic non-adhesive, lose surface mucins so that adhesion with the trophoblast can oc- mouse submandibular gland mesenchyme and embryonic mouse submandibu- cur. Recently, we have discovered that the heterodimeric sialomucin complex lar gland epithelium. The objective of this study was to examine the expression (SMC), which was first discovered in a mammary adenocarcinoma cell line, of an EGF-like ligand that was identified in the embryonic mouse submandibu- is present in a number of epithelia, including the uterus. The complex con- lar gland. A partial cDNA for an EGF-like gene was identified by RT-PCR sists of a mucin subunit and transmembrane protein which has two EGF-like utilizing degenerate EGF-like primers on embryonic mouse submandibular domains. Immunoblot and immunohistochemical analyses show that SMC is gland cDNA. An embryonic mouse cDNA library was screened with this par- apically expressed on the epithelium of virgin and early pregnant rat uterus. tial cDNA. Additional cDNA clones were isolated and sequenced. Homology Around midday of day 5 of pregnancy, SMC expression is down-regulated, search of nucleic acid databases indicated that these mouse cDNAs were ap- and the protein quickly disappears. Between days 8 and 10, SMC reappears. proximately 90% homologous to the rat Jagged cDNA. Jagged is a ligand for Sucrose-density show that SMC is expressed as a soluble form. In order to Notch (Cell, 1995, 80:909-917). Notch is a receptor for a phylogenetically determine whether SMC expression is hormonally regulated, in vivo exper- conserved signalling pathway that functions in cell-cell inductive interactions. iments using the antiestrogen tamoxifen were performed. After injection of Previous studies have localized mouse Notch expression to several embryonic tamoxifen into a pregnant rat at days 3, 4 and 5, SMC expression was still tissues including embryonic mouse submandibular gland epithelium (Develop- present at day 6, indicating that the down-regulation of SMC is dependent in ment, 1991, 113:199-205). Northern blot analysis indicated that mouse Jagged part on estrogen. Based on these data, we propose that SMC, secreted in a was expressed in the embryonic mouse submandibular gland as well as a vari- soluble form, helps to keep the uterus in a non-receptive state by binding to ety of other embryonic mouse tissues. Embryonic mouse submandibular gland and providing the epithelium non-adhesive properties. Then after pregnancy, mesenchyme was enzymatically separated from submandibular gland epithe- its disappearance at around day 5 allows interaction between trophoblast and lium. RT-PCR analysis indicated that Jagged was expressed in embryonic the uterine apical surface such that implantation proceeds. mouse submandibular gland mesenchyme. These results support a hypothesis that the Jagged-Notch signalling pathway participates in inductive signalling between embryonic salivary mesenchyme and embryonic salivary epithelium. Supported by NIH grant DE 10150.

648 649 OVEREXPRESSION OF CELL SURFACE FETAL SERTOLI CELLS DO NOT PREVENT MEIOSIS IN FEMALE 01,4-GALACTOSYLTRANSFERASE IN TRANSGENIC MICE GERM CELLS ((Norma Moreno-Mendoza and Horacio Merchant- PERTURBS MORPHOGENESIS IN MULTIPLE ORGAN SYSTEMS ((H. Larios)). Instituto de Investigaciones Biomedicas, UNAM. Apdo. Post. J. Hathaway and B. D. Shur.)) Department of Biochemistry and Molecular 70228, Mexico, D.F., Mexico 04510. Biology, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030 In the mouse fetal gonad, all germ cells start meiosis during fetal life in Cell surface /l1,4-galactosyltransferase (GalTase) mediates multiple cellular the ovary whereas in the testis meiosis begins later, in young pre-puberal interactions during fertilization and development, including sperm:egg recog- males. It was reported that ectopic mouse germ cells enter meciosis nition, intercellular adhesion, neural crest cell migration, and neurulation. regardless the sex of the fetus. This observation favored the view that Recent evidence suggests that surface GalTase performs these functions by germ cells in both sexes are intrinsically determined to enter meinosis serving as a signal transducing receptor. For example, interactions between unless they are prevented from doing so by Sertoli cells within the the cytoplasmic domain of GalTase and the actin-containing cytoskeleton fa- seminiferous cords (Zamboni and Upadhyay, 1983. J exp. Zool. cilitates cell migration on laminin matrices, and aggregation of sperm surface 228:173). Moreover, Burgoyne has suggested that the gene Sry may act GalTase induces the acrosome reaction by associating with heterotrimeric G as a trigger for Sertoli cell differentiation and that all other aspects of proteins. In both examples, altering levels of surface GalTase expression per- fetal testicular development are directed by these cells. Therefore, he turbs normal cellular behavior and subsequent downstream events. To further proposed that Sry may regulate the expresion of a factor for meiosis inhibition in the male fetus Phil. Trans. R. Soc. Lond. explore the effects of altered GalTase expression on development, we created (1988. 322:63). we have asked if the fetal Sertoli cells are able to inhibit the onset mice that overexpress GalTase at the cell surface. Several indepen- Here, transgenic of meiosis in both male and female cells. dent transgenic lines demonstrate common abnormalities, including a failure germ Morphologically undifferentiated gonads (11 dpc) from male and female fetus were to lactate and a high rate of neonatal lethality. Histological examination re- mixed and The was 6 in veals morphogenetic defects in several organs, including the mammary gland, dissociated, reaggregated. pellet kept days organ culture. It was found that most germ cells were incorporated together lung, liver, kidney, and gut. The affected tissues are characterized by a lack of, with Sertoli cells into the seminiferous cords. Although male germ cells or a reduction in, normal tissue organization, including abnormal cell-matrix associations, reduced cellularity, and hypervascularization. Primary cultures remained in the resting stage, female germ cells entered meiosis in established from transgenic tissues demonstrate abnormal cell-matrix inter- contact with Sertoli cells. This result demonstrate that female germ cells actions. Collectively, these results reinforce the notion that surface GalTase are early determined at the stage of morphologically undifferentiated gonad and, that Sertoli cells are unable to prevent the onset of meiosis in plays a key role in multiple cellular interactions during morphogenesis. Sup- ported by HD23479, HD22590, and DE07120 female germ cells. Supported by DGAPA-UNAM IN209694 112a Cell Interactions in Development (650-652). Sunday 650 651 CHANGES IN GAP JUNCTIONAL COMMUNICATION AND CONNEXIN EXPRESSION OF HEPATOCYTE cz MACROGLOBULIN BY EXPRESSION DURING MOUSE HAIR FOLLICLE DEVELOPMENT INTERACTION WITH ENDOTHELIAL CELLS IN CULTURE. (A. De ((R. Choudhry, J.D. Pitts and M.B. Hodgins)) Department ofDermatology, Maio, M. P. McCluskey, and M. A. Talamini)) Department of Surgery, University ofGlasgow, Glasgow G12 8QQ and Beatson Institute for Cancer Johns Hopkins University, School of Medicine, Baltimore, MD 21205. Research, Bearsden, Glasgow, G61 IBD, U.K. The liver is the central organ for synthesis of serum components and clearance of toxic substances. This organ is composed of different cell Hair follicles form in mouse skin between days 12 and 18 p.c. The embryo populations: hepatocytes, endothelial cells, Kupffer cells, and Ito cells. whisker pad produces large follicles that continue to develop normally when pads Although associations between these different cell-types is believed to play an are placed in culture. This provides an accessible system for studying changes in important role in liver function, little is known about the nature of the interactions among these cell-types within the organ. A co-culture system of gap junctional intercellular communication (GJIC) and connexin expression hepatocytes and endothelial cells was established and characterized. The during induction, bud formation, cell commitment, pattern formation and terminal major feature of this hepatocyte/endothelial cell co-culture was the presence of differentiation. GJIC was detected by micro injection ofLucifer Yellow CH (LY) a 180 kDa glycoprotein in the extracellular medium which was not detected in and connexin expression by indirect immunofluorescence. Lateral spread ofLY the extracellular medium of hepatocytes and endothelial cells grown was restricted in the epidermis at day 12 but increased within developing hair separately. This 180 kDa glycoprotein was identified as a2-macroglobulin by buds. LY spread was not seen between epidermal and dermal cells at any stage of sequencing proteolytic peptides derived from the purified protein. This finding was confmed by Northern and Western blotting analysis using follicle development. In the growing buds, extensive LY spread was associated probes specific for c2-macroglobulin. The expression of z2-macroglobulin with increased cell division and migration but the cells in the buds remained was sustained when hepatocytes interacted with endothelial cells from coupled to adjacent interfollicular epidermal keratinocytes. In the developing and different sources (aorta and pulmonary), skin fibroblasts, and an epithelial cell mature follicles LY spread throughout the bulb matrix and to cells showing early line of hepatic origin. However, interaction with macrophages or a kidney signs ofdifferentiation. Differentiation was associated with reduced LY spread, epithelial cell line did not support the expression of ot2-macroglobulin by restricted to compartments that correlated with different lineages. Tenninal hepatocytes. Expression of ce2-macroglobulin required direct contact between differentiation was associated with loss ofGJIC. Connexin 43 expression was hepatocytes and endothelial cells. Fixation ofendothelial cell monolayers allowed the attachment of hepatocytes but abolished the expression of ar mainly observed within the well coupled zones but connexin 26 was associated macroglobulin, suggesting that the role of endothelial cells is not just to with terminal differentiation and loss ofLY transfer. provide an attachment matrix. These findings suggest that endothelial cells may modulate hepatocyte gene expression by direct cell to cell contact

652 MICROGRAVITY-BASED THREE-DIMENSIONAL TRANSGENIC MODELS. (S.R. Gonda', T.C. Yang', R.C. Richmond', S. Provost', D.L. Wyborski?, W.S. Fitzgerald', C.D. Svrcek', K.A. George4, P.H. Fritz5)) 'NASA Johnson Space Center, Houston, TX; 'Dartmouth Medical School, Darthmouth College; 'Stratagene, LaJolla, CA; 4KRUG Life Sciences, Houston, TX; 'Universities Space Research, Houston, TX. (Spon. by Clarence F. Sams) The objective of this study is to develop and optimize microgravity-based three-dimensional tissue equivalent cell culture models with mammalian cells genetically engineered to include multiple copies of a defined target gene. Human mammary epithelial cells (H184B5) and Stratagene's Big Blue.' rat cell line Rat2l derived from the Rat2 embryonic fibroblast cells (ATCC CRL 1764), transfected with the pSV2NEO plasmid and cotransfected with Stratagene's Big Blue 45.5 kb shuttle vector, were cultured as mono- or co- cultures in the NASA developed bioreactor. Cells were inoculated atl to 5 x 10' celis/nl in DMEM with and without microcarriers and cultured for periods up to 21 days. Ninety percent of Rat2l cells cultured in the NASA bioreactor on Cytodex 3 beads at a cell/bead ratio of 20:1 attached and began to cover the beads by day 2. The cells covered the beads and formed large three-dimensional cell- bead aggregates by day 5. The addition of 2.4 x 105/ml of the human mammary epithelial cells to a day-five Rat2l fibroblast monoculture resulted in spheroidal-like cell structures after 8 days of coculture A 1:2 ratio of epithelial cells to fibroblasts was established by plating collagenase dissociated cells. No spheroid formation was observed controls. These results suggest that the NASA bioreactor supports the formation of three-dimensional aggregates of co-cultured transgenic cells. These three- dimensional tissue-like models will be highly useful for assessing environmental genotoxic agents and will provide the underpinning of general mechanisms of cellular organization and its importance in response to environmental insults. Growth Factors in Development I (653-654).

653 654 LYSOPHOSPHATIDIC ACID AND B-FGF MAINTAIN PROLIFERATING DIFFERENTIAL REGULATION OF MYOGENIC DIFFERENTIATION MYOBLASTS WITH DIFFERENT PROPERTIES ((S.Yoshida, A. OF RAT L6 MYOBLASTS BY EXOGENOUSLY ADDED AND Fujisawa-Sehara, K. Arai and Y. Nabeshima.)) Dept. of Molecular Genetics, ENDOGENOUSLY EXPRESSED FIBROBLAST GROWTH FACTOR-1. Natl. Inst. of Neuroscience, Natl. Ctr. of Neurology and Psychiatry, Tokyo, ((Takehito Uruno, Eiichi Mizukoshi, Kazuko Miyakawa, Junko Oki and Toru Japan (S.Y., A.F-S. and Y.N.) and Dept. of Molecular and Developmental Imamura.)) Cell Biology Laboratory, National Institute of Bioscience and Biology, Inst. of Medical Science, the Univ. of Tokyo, Tokyo, Japan (S.Y. Human Technology, Tsukuba, Ibaraki 305, Japan. and K.A.) Fibroblast growth factor(FGF)-l has been shown to repress differentiation Here we report that lysophosphatidic acid (LPA), a bioactive phospholipid, of myoblasts when added exogenously. As various myoblasts express FGF-1, promotes the growth and inhibits the differentiation of myoblasts in vitro in role of the endogenously expressed FGF-1 in the regulation of myogenesis is a distinct manner from basic fibroblast growth factor (bFGF). We revealed that subject of interest. We have cloned a highly differentiative L6 cell line (clone LPA stimulates the growth and, at the same time, inhibits the terminal dif- L6-10) from its low differentiative parent. The L6-10 cells spontaneously ex- ferentiation of the mouse C2C12 myoblasts. These effects were blocked by press elevated levels of myogenin gene, and readily express alpha-actin pro- pertussis toxin (PT), suggesting that LPA elicits these responses through Gi tein and form multinucleated myotubes after confluency. Interestingly, L6-10 proteins. Whereas bFGF, which activates receptor tyrosine kinases (RTK), transfectants overexpressing full-length FGF-1 in the cytosol still underwent promoted proliferation and inhibited differentiation in a PT insensitive man- differentiation. However, FGF-1 added to the culture of L6-10 cells repressed ner. We then found that LPA and bFGF act differently on the expression of all aspects of differentiation, although very little FGF receptors were found myogenic bHLH factors, the key players in myogenic differentiation: LPA stim- on the surface of the cells. A mutant FGF-l that lacks nuclear localization ulates the proliferation of undifferentiated myoblasts allowing the expression sequence also repressed the differentiation. When L6-10 cells were transfected of MyoD, while bFGF repressed its expression. Both compounds maintained to express kinase-negative FGF receptor, exogenously added FGF-1 did not myf-5 and suppressed myogenin. And, interestingly, while LPA did not inhibit repress their differentiation nor induce tyrosine phosphorylation of the cellular cell-cell contact-induced differentiation, bFGF strongly inhibited this process. proteins. The results indicate that endogenously expressed FGF-1 is not a re- These findings indicate that LPA and bFGF stimulate different sets of intra- pressor of myogenesis for L6-10 cells while exogenous FGF-1 potently repress cellular signaling pathways, resulting in proliferating myoblasts with distinct their differentiation through FGF receptor kinase. It was suggested that unlike myogenic bHLH factor expression and distinct differentiation potentials in re- mitogenic activity of FGF-l, differentiation-inhibiting activity of exogenously sponse to cell-cell contact. These illustrates the qualitative difference between added FGF-1 does not require nuclear localization signal of FGF-1. Gi-mediated and RTK-mediated mitogens. Sunday. Growth Factors in Development I (655-660) 113a

655 656 EFFECT OF BASIC FIBROBLAST GROWTH FACTOR (bFGF) ON C-MYC POST-NATAL GROWTH DEFICIENCY BY OVER-EXPRESSION OF EXPRESSION AND CRYSTALLIN SYNTHESIS DURING NEWT IRIS EPITHELIAL FIBROBLAST GROWTH FACTOR 9 (FGF9) IN TRANSGENIC MICE CELL (EEC) TRANSDIFFERENTIATION IN VITRO. ((C.L. Camacho, M. IAzaro, A. M. CHONDROCYTES. ((S. Garofalo, J. Cooke, M. Machado, G.P. Lunstrum, San Miguel, I. Garcia, P. Luper6n, V. Borero-Aldabondo*, L.D. Torres* and J.R. Ortiz)). A. Yayon* and W.A. Horton)) Shriners Hospital Research Unit - Oregon Dept. of Biology, UPR, Rfo Piedras Campus, San Juan, PR 00931-3360 and *Natnal Health Sciences University School of Medicine, Portland, Oregon and Sciences Dept., UPR-Carolina, PR 00984-4800. *Weizmann Institute of Science, Rehovot, Israel. (Spon. by W.A. Horton) The purpose of this research was to study the effect of bFGF and an antibody against it (Ab- are a bFCF) on the expression of c-myc protein and crystalline synthesis in vitro. Four-week IECs, FGFs family of nine polypeptides that can activate a family of four cultured in seruns-supplemented medium (SSM), were exposed for an additional week to either different tyrosine kinase receptor (FGFRI-4). They have important functions in serum-free medium (SFM), SSM plus bFOF or SSM plus Ab-bFPF, after which development, vascolarization and wound healing. Mutations in three FGFs immunocytochemical localization of the c-myc protein was performed. Results showed that c- receptors (FGFRI,2 and 3) have been identified in several human osteo- myc protein signal was not detected in nuclei of pigmented EECs cultured in SSM, although a chondrodysplasias. These mutations generate constitutively activated receptor. very weak signal was localized in cytoplasm. However, in depigmented lECs, the c-myc signal FGF9 (also know as Glial Activating Factor) was originally identified as a was strong in the cytoplasm, but weak in nuclei. Pigmented IECs, cultured in SFM, revealed a trophic growth factor for primary rat glial cells. It has ability to bind and activate weak c-myc signal in nuclei although in depigmented EECs a strong nuclear signal was FGFR2 and FGFR3. observed. In all EECs, under SFM conditions, almost no signal was observed in the cytoplasm. To understand the role of Fibroblast Growth Factors (FGF) in cartilage and Pigmented IECs, cultured in SSM plus bFOF, showed a very weak signal for c-myc protein in bone development we have generated gain of function transgenic mice over- both their nuclei and cytoplasm. A strong signal was observed in the cytoplasm of expressing FGF9 in chondrocytes. This was accomplished by joining the IECs, whereas a weak one was detected in nuclei. Pigmented as well as non pigmented cells, cultured in the presence of Ab-bFGF, showed a weak c-myc signal in nuclei chondrocytes-specific cis-regulatory elements of alpha 1(E1) procollagen gene to and no signal in cytoplasm. In other experiments, IECs were cultured for two-weeks in SSM mouse FGF9 cDNA. and transferred to either SSM plus bFGF or Ab-bFGF for an additional three weeks to analyze Three transgenic mice exhibit a phenotype characterized by growth deficiency of the synthesis of crystalline. Localization of crystallins was caWied out either with antibodies post-natal onset resulting in short limbs, spine and snout. The tail was kinky against whole lens proteins or anti-y-crystallin. IECs cultured in SSM plus bFGF showed a and the axial skeleton deformed. These mice died before weaning age. stronger crystallin signal than in controls without bFGF supplementation. No signal for Hystological analysis of cartilages shows a generalized disorganization of the crystallins was observed in cell cultures treated with Ab-FGF. Our results suggest that growth plate with very few hypertrophic chondrocytes. expression of c-myc protein seems to be regulated by growth factors, such as bFWF, and that c- The findings closely resemble those of human diseases due to constitutive myc could be an important regulator of EEC cell-cycle. Furthermore, bFGF may also play a activation of FGFR3. significant role in the control of crystallins synthesis. Therefore, both c-myc and bFGF seem to be important factors controlling IEC transdifferentiation. Supported by NIH MARC GRANT 5T34GM07821-16, NIH NIGMS/MBRSGRANT 08102-23. NIH Bridges GRANT R25GM48987-01. FIPI Office, UPR, Rio Piedras, and UPR-RCA CARP.

657 658 FGF-1 AND FGF-7 INDUCE DISTINCT PATTERNS OF GROWTH AND FIBROBLASTS INDUCE DIFFERENTIATION OF THE CRYPT-LIKE DIFFERENTIATION IN THE EMBRYONIC LUNG EPITHELIUM. ((W. V. EPITHELIAL CELL LINE T84 IN 3-DIMENSIONAL CO-CULTURE Cardoso, A. Ito*, H. Nogawace, I. Mason *** and J.S. Brody)). Pulmonary ((Halttunen T, Milki M.)) Institute of Medical Technology, University of Center, Boston Univ. School of Medicine, Boston, MA 02118;* Dept. of Tampere, Tampere, Finland. Medicine, Tenshi Hospital, Sapporo, 065;ceDept. of Biology, Chiba, Univ., The gut epithelium in crypt-villus axis represents a continuous developmen- Chiba 263, Japan;"*Div. Anatomy, Cell Biology, UMDS, London, SE19RT, UK. tal system. In the regulation of epithelial cell proliferation and differentiation Fibroblast growth factors (FGFs) and receptors (FGFR) are expressed the role of fibroblasts have been suggested to be fundamental. Our aim was in the developing lung and appear to be important regulators of lung growth to establish a co-culture method where these interactions can be studied. The and differentiation. Using mesenchyme-free lung epithelal cultures from day epithelial cells (T84) were cultured 3-dimensionally within type I collagen gel, on which the 11 embryonic mice, we show that FGF-1 (aFGF) and FGF-7 (KGF) produce fibroblasts (IMR-90) were allowed to grow as a monolayer with no straight different effects in the developing lung, likely by binding to FGFRs located at fibroblast-epithelial cell-to-cell contact. To study the effects of antag- onistic growth factors, namely transforming different sites In the lung epithelium. FGF-1 (100-500 ng/ml) stimulates lung growth factor-betal (TGF-betal) and hepatocyte growth factor (HGF), the fibroblasts were and either bud formation and induces sufactant protein SP-B mRNA expression at the omitted TGF-beta or HGF applied. In blocking studies antibodies against HGF or tips of the explant. FGF-7 (100-500 ng/ml) promotes uniform growth leading TGF-beta were added with fibroblasts. As result we found that HGF induced to formation of large cystic-like structures. In situ hybridization of FGFR-2 and significant increase in T84 proliferation rate, where as TGF-betal was respon- in day 11 lungs shows that FGFR-2 is uniformly distributed in the FGFR-4 sible for the organization into lumenal formations (81% of all colonies) and respiratory epithelium while FGFR-4 mRNA is restricted to the distal for enterocyte-like differentiation (49% of all epithelial cell colonies). When epithelium. We propose that the structures may a cystic-like represent the effect of TGF-betal in co-cultures was blocked by anti-TGF-beta, the generalized of these explants, from FGF-7 to a growth resulting binding colonies grow larger as in HGF-treated cultures. Furthermore, the HGF re- homogeneously distributed receptor, FGFR-2. However, bud formation likely ceptor c-met was abundantly present on crypt-like T84 cell colonies, but only may result from focal epithelial due to a high density of FGF-1 proliferation some if any c-met was detected on the basal surface of differentiated T84 ligandrecePtor intraion at t tips of the explants, likely FGFR-2 and FGFR- lumenal formations. The data suggests that balance between proliferation 4. FGF-7 also affects epihlial differentiation, restricting SP-C mRNA to a few and differentiation is regulated with TGF-betal. It inhibits proliferation by cells and inducing lamellar bodies formation in focal areas, as well as inducing down-regulating c-met with simultaneous induction of terminal differentiation both SP-A and SP-B mRNAs throughout the explant; changes not seen under pathway. FGF-1 treatment. Thus, different FGF members appear to play distinct roles in lung development.

659 660 EVOLUTIONARY CONSERVATION AND UNIQUENESS OF RECEPTOR THE ROLE OF THE EGF/TGF6K GROWTH FACTOR FAMILY IN THE BINDING SUGGEST THAT FGF-8b AND FGF-8f ARE FGF-8 PROTEIN DEVELOPMENT OF THE MALE UROGENITAL SYSTEM ISOFORMS IMPORTANT FOR VERTEBRATE DEVELOPMENT. ((J. ((Annemarie A. Donjacour, Peter Young and Gerald R. Cunha)) Department Gemell, A. Lawsh61, A. Blunt' D. M. Omitz3, and C. A. MacArthur' 2)) of Anatomy, UCSF, San Francisco CA 94143 Departments of Pediatrics', Pathology2, and Pharmacology and Molecular The of the male is Biology, Washington University School of Medicine, St. Louis, MO 63110. development urogenital (UG) system dependent upon androgens which act upon the unknown mechanisms to elicit (Spon. by J. Gemel.) mesenchyme by epithelial growth and morphogenesis. The EGF/TGFa family are possible growth factor mediators of this process. The role of the EGF/TGFa family in The murine Fgf8 gene encodes at least eight protein isoforms by alternative the male UG tract was examined in two lines of transgenic mice: a TGFa splicing of multiple 5' exons. We have produced seven of the known FGF-8 knock-out (KO) and an EGFR KO. Neonatal seminal vesicles (SV) and protein isoforms by recombinant methods with carboxyl-terminal histidine ventral prostates (VP) were photographed and the areas of the glands tags, and have analyzed their abilities to stimulate mitogenesis in cell lines calculated. Some organs were grafted under the renal capsule of male mice, containing defined solitary FGF receptors. FGF-8b and FGF-8f are unique grown for 3 mo and weighed. TGFa KO males were normal in size and all The EGFR KO mice among FGF-8 protein isoforms in that they stimulate mitogenesis in cells of their UG organs grew normally. grew poorly, rarely containing FGFR2c. We have cloned the human FGF8 gene, and observed surviving past 10 days postnatal, and were smaller than wild-type (wt) litter mates. However, the area of the male UG while smaller, were that most of the coding sequences are highly conserved between mice and organs, similar to their wt litter mates when corrected for body weight (3.9 KO SV, 4.3 humans. Surprisingly, the longer form of exon1 B is not conserved between wt SV, 1.2 KO VP, 1.5 wt VP x105 ±em2/ gm body weight, 6d postnatal). When mice and humans, and the human gene contains two in-frame stop codons neonatal SV or VP were grown as grafts, EGFR KO glands attained the same in this region. the gene four protein Therefore, human encodes only weight as those from wt mice (279±39 KO SV, 363±78 wt SV, 14±8 KO VP, 18±6 to and KO isoforms (FGF-8a, FGF-8b, FGF-8e, FGF-8f). The unique ability wt VP mg wet weight ± SE). Secretory proteins were made by both wt stimulate mitogenesis from FGFR2c binding and the strong evolutionary grafts. In contrast in serum-free organ culture, growth and morphogenesis of conservation between mice and humans suggest that FGF-8b and FGF-8f normal neonatal SV stimulated by testosterone (T) was reversibly blocked by are FGF-8 protein isoforms important for vertebrate development. two antisera raised against TGFa. SV from the EGFR KO and TGFa KO mice also grew in the presence of T, and were unexpectedly blocked in their development by antisera to TGFa. These data suggest that EGF/TGFa- related ligands, possibly those that bind to the Her2, 3 or 4 receptors may be able to compensate for the lack of EGFR or TGFa in the male UG system. 114a Growth Factors in Development I (661-666). Sunday

661 662 INACTIVATION OF THE EGF-R FUNCTION RESULTS IN A MODULATION OF SURFACTANT PROTEIN GENE EXPRESSION DURING B-CELL MIGRATION DEFECT ((Miettinen PJ, Ustinov J, EARLYMOUSEEMBRYONICLUNGFORMATIONBYEPIDERMALGROWTH PANCREATIC (EGF). ((Dimitrios Hatzis and Heber C. Nielsen)) FloatingHospital for M, Koivisto Cirulli V@, Werb ZF+, Otonkoski T.)) University of FACTOR The Huotari T, Tufts School of Medicine, Boston, MA 02111. Helsinki, UCSD@ and UCSF+ Children, University branching morphogenesis. However, little is The pancreas forms through During early embryonic lung branching morphogenesis (BM) the changing pattern of about the regulators of this process. We have previously shown that known surfactant protein (SP) gene expression reflects the progression of epithelial the developing pancreas while its ligand TGF- EGF-R is expressed throughout differentiation. SP-C expression begins in both proximal and distal epithelium on day a is in the epithelial compartment and together with insulin in the localized ell and becomes restricted to distal epithelium. SP-A expression begins on e14 in This suggests a /3-cell-specific action for TGF-o during development. b-cells. proximal airway epithelium as SP-C expression is lost in these cells. We and others into the developmental and physiological role of EGF-R me- To gain insight have shown that EGF stimulates and transforming growth factor beta-1 (TGFP 1) diated signalling in pancreas, we analyzed the pancreatic phenotype of the regulate mice die P8 from inhibits BM. We hypothesized that growth factors which influence BM also EGF-R deficient mice. As described previously, these by embryonic lung epithelial differentiation, and studied this using quantitative-competitive For the pancreatic studies we used newborn pups de- epithelial immaturity. PCR to measure SP-A and SP-C gene expression in mouse embryonic lung cultures. intercrosses (n=3D7 for EGF- R-/- and n=3D6 for riving from EGF-R+/- Embryonic lungs (day elO.5) were cultured in serum-free medium for 72 hrs with: 1) control pups). As expected, the general morphology of the EGF-R-/- pancre- 1 (2ng/ml). no exogenous growth factors (control), 2) EGF(l0ng/ml), and 3) TGFP structure was not as as ata appeared normal. However, the acinar organized Total RNA was harvested and reverse transcribed into cDNA. SP-A and SP-C specific more of a E17 in the newborn controls, but resembled the immature structure primers (spanning the smallest intron for each gene) were used to amplify the cells wild-type pancreata. Furthermore, proliferation of pancreatic epithelial respective cDNA segments. Whole mouse genomic DNA was used as competitor. cell types were present. was only 50% of that in controls. All pancreatic islet Restriction endonuclease analysis verified PCR product identity. SP-A expression was the islet cells were located However, instead of forming circular clusters mainly not detected under any condition. SP-C expression was increased 6-fold by EGF with ducts. A morpho- in streak-like structures directly associated pancreatic to or 1 did not alter SP-C expression. islets compared control TGFP 1-treated cultures. TGFO metric analysis revealed that the proportion of /3-cells in duct-associated In conclusion, EGF simultaneously stimulates BM and development of the distal was 64±8% in the EGF-R-/- pancreata as compared with 36 ± 9% in controls epithelial phenotype. We speculate that regulation ofBM by EGF is associated with (p<0.05). Our findings suggest that the absence of EGF-R mediated signalling modulation of lung epithelial cell differentiation. leads to defective migration of the pancreatic /3-cell as they differentiate from ductal precursor cells.

663 664 HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR EXPRESSION IN HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR ALTERS LIVER GROWTH AND DEVELOPMENT IN TRANSGENIC MICE Song and Paul G. McGuire)) Dept. of THE DEVELOPING HEART ((Wanmin ((H. Sakata, H. Takayama, R. Sharp, J. S. Rubin, G. Merlino and W. J. Anatomy, University of New Mexico School of Medicine, Albuquerque, NM LaRochelle )) NCI, Bethesda, MD 20892. (spon. by G. Merlino) 87131 Since its initial detection as a hormone-like activity that stimulated

hepatocyte proliferation, HGF/SF was also unexpectedly discovered to a The endocardium of the developing cushion tissues undergoes motogenic and morphogenic properties. To investigate the in vim role of HGF/SF transformation into mesenchymal cells which rapidly invade the surrounding in liver function, we generated transgenic mice using HGF/SF cDNA under extracellular matrix. This process is essential for the normal formation of the the control of the mouse metallothionein gene promoter and 5'/3' flanking valves and specific portions of the atrial and ventricular septum. Several sequences. In adult HGF/SF transgenic mice, liver weight percentage of total factors have been identified which appear to initiate and control this body weight was at least twice that of wild-type mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and transformation process including the growth factor TGFB3 and the matrix- appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. factors including Hepatocyte Growth associated ES antigens. Other additional Strikingly, transgenic livers exhibited a significant increase in the number of

Factor / Scatter Factor may also be involved in the initiation of this process as small hepatocytes with a 2N DNA content. The DNA labeling index of enlargement well as the maintenance of the mesenchymal phenotype. The activated form of hepatocytes increased eleven-fold at four weeks of age, when liver Hepatocyte Growth Factor/Scatter factor binds to the c-met proto-oncogene in first became apparent, and was still elevated almost five-fold in adult HGF/SF responsive cells and stimulates both mitogenic and motogenic behaviors. transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every two days in culture while little growth observed with Using an immunochemical approach, we have confirmed the presence of control hepatocytes. Met and substrates such PI3-kinase, Shc, pp60c-src, HGF/Scatter Factor in the developing heart and have localized its production and paxillin were activated in hepatocyte cell line established from to the myocardium of the atrioventricular canal and outflow tract. Isolated p125FAK, HGF/SF transgenic liver. Although no hepatocellular carcinomas found, are to the growth factor and the activated form of of endocardial cells responsive several unusual tumors of sarcoma origin were revealed. Finally, the rate a the livers the protein is present in the developing heart suggesting role in transgenic mouse liver regeneration was increased three-fold over control regulation of mesenchyme migration and proliferation following partial hepatectomy. Together, these data indicate that HGF/SF functions as a potent stimulus of hepatic proliferation and regeneration when overexpressed in vivo, and strongly suggest that HGF/SF plays prominent role in regulating normal liver growth, repair, and development.

665 666 PROSTAGLANDIN El AND SERUM MODULATE MADIN DARBY CANINE KIDNEY INTERACTION OF GP75 AND TRKA NERVE GROWTH FACTOR CELL RESPONSES TO HEPATOCYTE GROW1H FACTOR. ((S.A. Orellana, D.J. RECEPTORS. ((David E. WolW Alonzo R Ross, Christine McKinnon, Marie-Claire Winkler, & E.D. Avner)), Department of Pediatrics, Case Western Reserve University and Daou, Robert M. Stephens and David R Kaplan)) Worcester Foundation for OH 44106. Rainbow Babies & Children's Hospital, Cleveland, Biomedical Research, Shrewsbury, MA 01545, Frederick Cancer Center, Frederick, MD 21701, Montreal Neurological Institute, Montreal, Quebec, Canada H3A 2B4. Discrete mechanisms may mediate morphogenesis in developing kidney. Roles for tyrosine kinase and cyclic AMP-dependent protein kinase in tubule and cyst formation were proposed for 4 Madin Darby Canine Kidney (MDCK) cell clones (designated by #s The nerve growth factor receptor plays a critical role in neuronal development, and kinase 22,25,28, and 29) that display deferential responsiveness to morphogens maintenance, and repair. Neurotrophic factor receptors can enhance neuronal El (PGEI). To determine if activators hepatocyte growth factor (HGF) and prostaglandin survival and, thereby, reduce neurodegeneration resulting from neuropathies and HGF sensitivities were responsible for diferences in clonal cyst and tubule formation, gel aging. There two receptors for NGF, gp75 and the tyrosine kinase TrkA. It has cultures were overlaid with defined medium without PGE1, defined medium with 7OnM of PGEI (PGE), or medium plus 5% fetal bovine serum (FBS). Over a range of recombinant been hypothesized that the high affinity receptor is a heteromolecular complex human HGF concentrations (1-100ng/ml). incidence of specific structures was recorded. gp75 and TrkA. Using fluorescence recovery after photobleaching (FRAP) and in these clones. Increased of Process formation (PR) predicts tubule formation incidence copatching confocal microscopy we have demonstrated the existence of this PR in HGF-responsive clones 22 and 28 occured with a 112max HGF dose of 5ng/ml in complex. Gp75 complexes with TrkA but not the BDNF receptor TrkB, PDGF FBS. In FBS, HGF at all doses tested failed to increase PR in unresponsive clones 25 or torso. Employing chimeras of TrkA and torso we have demonstrated and 29. In PGE, the range of HGF doses failed to induce PR in any clone. In PGE. receptor, spontaneous PR was inhibited in all clones and cyst incidence (CY) was increased in 22. that interactions between the extracellular domains of gp75 and TrkA dominate their 25, and 29. In 22 only, HGF added to PGE decreased CY and increased incidence of interaction. FRAP measurements of TrkA diffusion show that coexpression with response of In 22 in CY and tumor-like (iT) structures with the same 1/2max 1Ong/ml. FBS, gp75 does not immobilize TrkA. NGF immobilizes TrkA both in the presence the dose appeared to be also declined as HGF dose increased, but 1/2max HGF

667 668 LIGAND MULTIMERIZATION: A SWITCH FOR ENDOTHELIAL ASSEMBLY The developing renal vascular system: analysis of its spatial organization OF CAPILLARY-LIKE STRUCTURES THROUGH THE EPH-LIKE RECEPTOR, and modulation by angiogenic factors. ((Sabine Kloth, Claudia Ebenbeck, ELK. ((Elke Stein@, Harald O.Schoecklmann, Douglas Pat Cerretti", and Thomas 0. Jan Monzer, Will W. Minuth.)) Institute of Anatomy, University of Daniel )) Departments of OPharmacology, Medicine and Cell Biology, Vanderbilt Regensburg, D-93053 Regensburg, Germany TN 37232, University School of Medicine, Nashville, Universitat Numbergn The function of the kidney depends on coordinated interaction of different GERMANY and "Immunex Corp., Seattle, WA 98101. tissue components. Blood filtration in the renal glomeruli and further filtrate processing in the nephron are based on an undisturbed blood supply of the The Eph-family receptor, ELK, was previously identified in the central nervous kidney. The coordinated development of the vascular system and the nephrons to and specification, lire, we system where it is anticipated signal neuronal targeting is a prerequisite for proper organ function. In our study on kidney vessel de- expression A report ELK and discriminatory function in human endothelial cells. velopment we raised endothelium detecting antibodies that detect different predominant 4.4 kb ELK mRNA transcript was identified in cultured renal developmental stages of endothelial cells. With these antibodies an extraordi- microvascular and umbilical vein endothelial cells. A soluble, dimeric Fc fusion form narily high degree of spatial organization was detected in the developing renal of the transk membrane ELK ligand, LERK-2, stimulated phosphorylation of tyrosine microvasculature. The three-dimensional structure of the vascular system was endothelial ELK receptors at concentrations. Striking differences were subnanomolar analyzed by laser scan microscopy. A model of the developing vessel system in effects of LERK-2/Fc to promote microvascular endothelial cell assembly observed was reconstructed from serial optical sections. In another set of experiments, into capillary-like cord and tube structures, depending upon its state of explants of the nephrogenic zone were cultured under continuous medium per- multimerization with an anti-Fc antibody, assembly required antibody clustering of fusion. Perfusion allowed complete omission of serum in the culture media. ligand, but was independent of effects of either antibody or Fc fusion domain. A To analyze the influence of angiogenic factors and hormones on vascular dif- biochemical correlate for this observation was identified as clustered, but not ferentiation, the medium was supplemented with aldosterone and vitamin D3, unclustered, LERK-2/Fc promoted co-immunoprecipitation of a 16 kDa tyrosine vascular endothelial growth factor (VEGF) or basic fibroblast growth factor phosphoprotein with activated ELK (bFGF).The addition of VEGF or the hormones resulted in excellent preser- presented to ELK We conclude that the multimeric state of ligand endothelial vation of the spatial structure of the vascular network. However, endothelial receptors provides a differentiable signal that impacts cellular responses and tyrosine cells were not detectable in explants cultured in the presence of bFGF. phosphorylation of distinct intracellular substrates. These results identify ELK ligand multimerization as a switch that determines endothelial assembly responses and suggest a paradigm for cellular aggregation responses mediated through Eph family receptors.

669 670 DISRUPTION OF NEUREGULIN, ERBB2 AND ERBB3 GENES: FOLLICLE STIMULATING HORMONE INDUCES TERMINAL CARDIAC AND NEURAL ABNORMALITIES ILLUSTRATE ROLES IN DIFFERENTIATION IN A PREDIFFERENTIATED RAT DEVELOPMENT. ((S.L. Erickson, K.S. O'Shea*, and M.W. Moore)) GRANULOSA CELL LINE (ROG).(( Ronghao Li1, Alison Department of Molecular Biology, Moore1, David Genentech Inc., South San Francisco, California, 94080. * Department of M. Phillips2, Jennie P. Matherl)).lCell Biology, Discovery Research, Genentech, Anatomy and Cell Biology, University of Michigan, Ann Arbor, Michigan, Inc. 460 Point San Bruno Boulevard, So. San Francisco, CA 94080. 2The 48109 Population Council, 1230 York Avenue, New York.

The neuregulins are a single-gene family of ligands (heregulins, neu Granulosa cells are the nurse cells for the developing oocytes in mammalian ovaries. differentiation factors) that bind to ErbB2 in conjunction with ErbB3 and The divergent commitment of granulosa cells to proliferation or differentiation and ErbB4. Overexpression of the ErbB2 protein in human cancers such as programmed cell death directly reflects the selection of follicular dominance and breast and ovarian carcinomas, is associated with increased invasiveness atresia. This process is largely regulated by pituitary follicle stimulating hormone and decreased responsiveness to current treatments. Mice were generated (FSH) and local autocrine and paracrine factors such as activin (R. Li, et al. 1985 lacking either the entire neuregulin family of alternatively spliced ligands, the Endocrinol. 136:849). In order to further analyze the role of FSH and intra-ovarian ErbB2 receptor or the ErbB3 receptor. NeureguIin and ErbB2 null factors in follicular selection, we have established a rat ovarian granulosa cell line homozygous embryos die at E10.5 on both 129 X C57BL/6 and 129 X (ROG) from day 14 rat ovary. The cells have been continuously carried in serum free, Balb/c backgrounds. They exhibit cranial ganglia abnormalities and a lack of defined medium in the presence of recombinant human activin A but without FSH, cardiac ventricular trabeculation. ErbB3 null homozygous embryos however, for over 2 years. The cells maintain their FSH receptors and respond to FSH by a die at El 3.5 on both strains. In addition to anomalous development of otic burst of increased cell proliferation, and then cease dividing. This result supports vesicles and cardiac cushions, the cerebellum is hypoplastic. These the hypothesis that activin is a key factor regulating the proliferation of granulosa observations correlate with known cardiac expression patterns indicating that cells and the maintenance of immature granulosa cell properties. In addition, neuregulin and ErbB2 play a crucial role in ventricular trabeculation whereas withdrawal of FSH from FSH-primed ROG cell cultures results in massive apoptotic ErbB3 is critical in valve development. Neuregulin, ErbB2 and ErbB3 are all cell death, confirmed by detection of intemucleosome DNA fragmentation. Time critical in neural crest migration leading to cranial ganglia formation and ErbB3 lapse recording reveals ROG cells start active membrane blebbing at two hours after is shown to be intimately involved in cerebellar and otic vesicle FSH withdrawal and most cells die within 7 hours. SEM micrographs show that FSH development. primed ROG cells have numerous microvilli on the cell surface. Complete disappearance of these microvilli occurs by 2 hours after FSH withdrawal , concurrent with the onset of apoptosis. This data indicates that FSH induces the ROG cell line to differentiate irreversibly into a more mature granulosa cell whose survival is dependent on FSH. The ROG cell line thus provides a good model of follicular selection.

671 672 EXPRESSION OF ARACHIDONIC ACID EPOXYGENASE CYP2J3 NON-CATALYTIC TRK B RECEPTORS ARE EXPRESSED IN DURING HEART DEVELOPMENT: ANGIOGENIC RELATIONSHIP. DEVELOPING NON-NEURONAL TISSUES IN PATTERNS THAT ((G.L. Engelmann and D.C. Zeldin*)) Dept. of Medicine & Cell Biology, INDICATE THE RECEPTOR REGULATES TARGET-DERIVED Loyola University, Maywood, IL; *NIH/NIEHS, Res. Triangle Pk., NC. NEUROTROPHIN CONCENTRATIONS. ((E.F. Wheeler, H. Gong, N. Gracia, R. Grimes, L. Garcia, D. J. Stuart Division of of the heart its during Benoit, L.Vasquez, )) Growth mammalian and cellular constituents Life Sciences, The University of Texas at San San Antonio, TX postnatal development are tightly is Antonio, regulated. Neonatal development 78249 a transition period of proliferative-to-non-proliferative myocytes in concert with active ventricular remodeling. We have shown rat ven- tricles to be sources of protein & transcripts for CYP2J3, a cytochrome The expression of the non-catalytictrk B receptor was examined in rat embryos P-450 arachidonic acid epoxygenase highly expressed in myocytes. by quantitative in situ hybridization and was observed to vary in non-neural Furthermore, CYP2J3 products, cis- epoxyeicosatrienoic acids, (EETs), tissues surrounding the developing vibrissae, muscle, cochlea and olfactory are endogenous to the heart. Northern- blot hybridization analyses epithelium. Quantitative morphometric analysis was used to measure the levels show CYP2J3 transcripts to be transiently upregulated during postna- of expression of thetrk B receptor in the tissues surrounding the developing tal development between weeks 3-9 of age when activecapillary muscle and vibrissae. Increases in non-catalytictrk B receptor expression in the angiogenesis is known to occur. Western blot analyses indicate that mesenchymal cells of the maxillary pad occur during stages of development are detected as cardiac microsomal levels of CYP2J3 protein readily when the trigeminal neurons undergo programmed cell death. Similarly, of active early as 1-day postbirth; yet also increased during periods increased non-catalytictrk B expression levels in tissues muscle angiogenesis. Notably, CYP2J3protein levels markedly increased in surrounding microsomes from 5-to 7-week old spontaneously hypertensive rats correlate with the onset and progression of motor neuron cell death. The spatial (SHR). Therefore, myocyte-derived EETs may facilitate/ modulate temporal patterns of expression of the non-catalytictrk B receptor indicate it may serve concentrations capillary angiogenesis; perhaps via the vascular endothelial growth as a buffer that confines and regulates neurotrophin factor (VEGF) family of ligands and receptors. Importantly, EET- within target tissues. To support this hypothesis, the ability of the non-catalytic treated mvocvte cell lines increase steady-state mRNA levels and syn- receptor to bind and internalize factor was tested in vitro. CHO cells were thesis of VEGF, a potent, myocyte-derived paracrine angiogemc actor, transfected with the non-catalytic form ofthetrk B receptor (TI) lines to that of the full into conditioned media. Rat myocyte-cell stably over-expressing binding and internalization was compared length trk will be used to evaluate this and in vitro studies the human homologue CYP2J2 critically Taken together, the results of the in vivo potential VEGF A the temporal, receptor. relationship. paradigm illustrating that the functions in non-neural cells to sequester correlative developmental changes in expression of CYP2J3 and VEGF non-catalytic receptor that diffuses away from target tissues. Such a mechanism would or VEGF-receptors with myocyte proliferation and capillary angiogenesis during heart development will be presented. Supported, neurons remain directed toward the neurotrophins that stay confined This work was the San Antonio Area Foundation, in part, by HL-42218 (GLE) & Div. of Intramural Res. (DCZ). target tissue. supported by NIH MBRS 2 806 GM08194-16 & NIH MARC GM077-17. 116a Growth Factors in Development I (673-675). Sunday

673 674 NEWT MYOTUBES RE-ENTER THE CELL CYCLE ON SERUM MODULATION OF C-RET AND GDNF EXPRESSION IN RAT STIMULATION.((E.M. Tanaka, A.A.F. Gann, P.B. Gates and J.P. METANEPHROS ORGAN CULTURE (MOC) BY RETINOIC ACID. ((T. Brockes*))Ludwig Institute for Cancer Research, and *Dept. Biochemistry Gilbert, J. Vilar, E. Moreau and C. Merlet-Benichou.)) INSERM U.319, and Molecular Biology, University College London, 91 Riding House University Paris 7-Denis Diderot, 75251 Paris, France. Street, London WIP 8BT ENGLAND Using an in vitro model of renal differentiation, MOC, we previously showed that retinoids are dose-dependent stimulators of nephrogenesis. Here we in- In the regenerating newt limb, multinucleate skeletal muscle can reverse its vestigated the effects of retinoic acid (RA) on the expression of the tyrosine- differentiation to generate the proliferating, mononucleate cells that are the kinase membrane receptor encoded by the proto-oncogene c-ret, and of the progenitor cells for regeneration. We have been studying this process in glial cell-line derived neurotrophic factor (GDNF), both molecules playing vitro using cultured newt myotubes. These myotubes appear to differentiate a role in epithelium-mesenchyme interactions during kidney development. fully; withdrawing from the cell cycle, and expressing muscle-specific Metanephroi from 14 days rat embryos were cultured for up to 6d, and for each myosin. Unlike mammalian myotubes, however, they re-enter S-phase and embryo, one metanephros was grown in a serum-free medium as control and then arrest in G2 when re-stimulated with serum. This property is strikingly the controlateral in the same medium with 100 nM RA, a concentration that similar to myogenic cells taken from Rb-/Rb- knockout transgenic mice. stimulates in vitro nephrogenesis by 3-fold. Western n blot analyses showed Differences in the regulation of Rb could underlie the special properties of that at the time of explantation, c-ret exhibits 3 highly ly expressed isoforms, the newt cells. We have cloned the newt Rb gene, which is 62% similar to and GDNF exhibits a strong signal. During MOC development, expression the human Rb gene. The gene is expressed in these cells indicating that the of both markers decreases rapidly, despite continuous meta-nephros growth. cells are not null for Rb. We have shown that phosphorylation of Rb is The presence of RA in the culture medium maintains high expression of c-ret required for the cell cycle re-entry. Forced expression of human pl6INK4 during 4d, but the expression of GDNF was lower than in controls after 48h in the newt myotubes completely inhibits the serum response. p16 inhibits of culture. This decrease of endogenous GDNF pool is consistent with the the cell cycle kinase CDK4/6 whose only substrate is thought to be Rb. recent proposal that this factor might be a potential ligand for c-ret receptor. Furthermore, transfection of newt myotubes with a mutant Rb, A34, which Immunocytochemical studies are in progress to precise where these changes lacks all consensus CDK phosphorylation sites inhibits cell cycle re-entry by have taken place. These data indicate that the stimulating effect of RA on 91% compared with 28% inhibition when wild-type Rb is overexpressed. branching morphogenesis and nephrogenesis in MOC is likely to be mediated We are also initiating studies to identify the stimulatory factor in by expression level of the c- ret proto-oncogene. This highlights the role played serum. It is not one of a host of common growth factors or extracellular by retinoic acid in renal organogenesis. matrix molecules tested thus far. It appears to be a protein of approximately 40 kD which associates with a thrombin-rich protein complex.

675 ANALYSIS OF STEEL MUTANT EMBRYOS REVEAL THAT MELANOCYTE PRECURSOR MIGRATION IS DIRECTED BY STEEL FACTOR ((B. Wehrle-Haller and J.A. Weston.)) Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254 In the mouse, melanocyte precursors (MPs) expressing the receptor tyrosine kinase c-kit, appear lateral to the neural tube and migrate to the dermatome, and subsequently the epidermis, where mRNA for Steel factor (SLF; ligand of c-kit) is expressed. SLF is present in a secreted and membrane bound form at the basolateral surface of the dermatome facing the MP migration pathway and is later produced by keratinocytes. In embryos lacking SLF, MPs appear but fail to migrate and ultimately disappear. This illustrates the requirement of SLF activity for migration and survival of c-kit expressing MPs. Mutations that alter the presentation of SLF activity in the tissue may therefore affect the migration and localization of MPs. Such mutations include the Steel (So alleles SI' and 51P7H. SId is a deletion of the transmembrane and the cytoplasmic domain of SLF resulting in the production of constitutively secreted SLF. Sid mutant protein is initially present but quickly disappears from the lateral pathway. Loss of SLF correlates with the disappearance of MPs initially present on the lateral pathway. This indicates, that membrane anchored SLF is required for survival of MPs on the lateral pathway. SP7H is a mutation that exhibits an altered cytoplasmic domain of SLF. In SP7H mutant embryos MPs survive on the lateral pathway, but fail to enter the epidermis. Thus, we postulate that the cytoplasmic tail of SLF controls presentation of SLF required for MP migration into the epidermis, possibly by providing basolateral targeting signals in polarized SLF producing cells. Supported by NIH DE04316.

Cell Polarity (676-677).

676 677 THE FUNCTIONALLY REDUNDANT MIP23 AND MPH23 GENES DIFFERENTIAL DISTRIBUTION OF CDC42 DURING THE ARE REQUIRED FOR POLARIZED CELL GROWTH IN YEAST. DEVELOPMENT OF EPITHELIAL POLARITY ((Ruth Kroschewski and ((G.-C. Chen, and C.S.M. Chan)) Department of Microbiology, Ira Mellman.)) Yale University, School of Medicine, Dept. Cell Biology, New Haven, CT 06520-8002, USA University of Texas, Austin, TX 78712. (Spon. by C.S.M. Chan.) CDC42 is a small GTPase that is essential for polarized budding in yeast. While its mechanism of action is unknown, CDC42 is involved in cytoskeletal The BEM2 gene of the budding yeast Saccharomyces cerevisiae rearrangements and signaling cascades. Because CDC42 in highly conserved, encodes a Rho-type GTPase-activating protein that is required for it may be required for the establishment of polarity in epithelial cells.To ad- proper bud-site selection at 26°C and bud emergence at 37°C. We dress this question we are developing a single cell polarization assay using have identified MIP23 as a gene that, when present in high copy the MDCK epithelial cell. The distribution of apical and basolateral plasma number, can suppress partially the bud-site selection and bud membrane proteins was determined by immunofluorescence at various times emergence defects of bem2 mutant cells. The Mip23 protein is after cell-substrate attachment. Analysis of xz sections of cells revealed that plasma membrane polarity was induced shortly after attachment. We next closely related in sequence (39% identity) to the Mph23 protein. asked whether the development of polarity was accomplished by alterations or no phenotype, Yeast cells lacking Mip23 Mph23 have discernable in the distribution of CDC42. Early after plating of single MDCK cells en- whereas cells lacking both proteins are defective in bud emergence dogenous CDC42 localizes cytosolic and associated with or near the plasma at high temperature (i.e., similar to bem2 mutant phenotype). membrane. Several days later, a more dramatic alteration of the localization Mip23 and Mph23 each contains a short stretch of sequence that is of CDC42 was observed. In fully polarized MDCK cells, CDC42 was found partially homologous to a sequence motif found in proteins that in discrete dots in the perinuclear region and no longer in the vicinity of the bind the Cdc42 (and other mammalian Rho- plasma membrane. Thus CDC42 cycles between different localization stages yeast Rho-type GTPase during the epithelial polarity development. type GTIases). Interestingly, the temperature-sensitive phenotype of mip23 mph23 double mutant cells can be suppressed by the overproduction of the Cdc42 or Rsrl/Budl GTPase. Furthermore, mutations in MPH23, but not MIP23, exacerbates the cdc42 mutant phenotypes. These results together suggest that Mip23 and Mph23 function in the same morphogenetic pathway as Bem2, possibly through their interaction with Cdc42 or Rsrl. Sunday. Cell Polarity (678-683) 117a

678 679 ROLES FOR RAC1 AND CDC42 IN PLANAR POLARIZATION AND ROLES OF THE MEMBRANE CYTOSKELETON AND CADHERIN- HAIR FORMATION IN THE WING OF DROSOPHILA ((S. Eaton, R. MEDIATED CELL-CELL ADHESION IN GENERATING EPITHELIAL Wepf, and K. Simons.)) Programme in Cell BiologyEMBL POLARITY IN VIVO. ((P.A. Piepenhagen and W.J. Nelson)) The wing of Drosophila melanogaster is covered by an array of distally Department of Molecular and Cellular Physiology, Stanford pointing hairs. A hair begins as a single membrane outgrowth from each wing University School of Medicine, Stanford, CA 94305. epithelial cell, and its distal orientation is determined by the restriction of outgrowth to a single distal site on the cell circumference. We have examined Studies using cultured renal epithelial cells indicate that the the roles of Cdc42 and Racl in the formation of wing hairs. We find that basal-lateral distribution of Na/K-ATPase is regulated by Cdc42 is required for localized actin polymerization in the extending wing interactions with the membrane cytoskeleton and E-cadherin- hair. Interfering with Cdc42 activity by expression of a dominant negative mediated adherens junctions. Previously, we examined the protein abolishes both localized actin polymerization and hair outgrowth. In relevance of these In vitro observations to the maintenance of contrast, Racl is important for restricting the site at which hairs grow out. epithelial polarity in vivo using the mouse nephron as a model Cells expressing the dominant negative RaclN17 fail to restrict outgrowth system (Piepenhagen et al. 1995. Am. J. Physiol. 269: C1417-C1432). were to a single site and give rise to multiple wing hairs. This polarity defect Our results consistent with E-cadherin providing positional to membrane is associated with disturbances in the organization of junctional actin, and information organize the cytoskeleton and indicated a mechanism for restricting also with disruption of an intricate microtubule network that is intimately that Na/K-ATPae subcellular distributions through interactions with the associated with the junctional region. We also find that apical junctions and membrane cytoskeleton is likely to be relevant in vivo. We have microtubules are involved in structural aspects of hair outgrowth. During hair now extended these studies to the developing mouse kidney. Using formation, the apical microtubules that point distally elongate and fill the detergent fractionation and quantitative immunoblotting, we emerging wing hair. As the hair elongates, junctional proteins are reorganized have examined the expression of Na/K-ATPase, ankyrin, fodrin, on the proximal and distal edges of each cell. and E-cadherin beginning at embryonic day 12 (E12) and continuing on through adulthood. Our data indicate that a rapid increase in ankyrin expression correlates both with the accumulation of Na/K-ATPase during kidney development and with the development of Triton X-100 insolubility of Na/K-ATPase and E-cadherin. These data suggest that ankyrin may be a rate limiting factor in the assembly of a Triton X-100 resistant membrane cytoskeletal structure which stabilizes Na/K-ATPase.

680 681 ACTIN BINDING PROTEINS DEFINE DIFFERENT ASYMMETRIES IN THE INTERDEPENDENCE OF CURVATURE AND THE DISTRIBUTION OF MEMBRANE DEVELOPMENT OF THE EARLY C. ELEGANS EMBRYO. ((R.V. Aroian, C. Field, PROTEINS MAY FUNCTION AS THE PHYSICAL BASIS OF CELL POLARITY. and B. Alberts)) Department of Biochemistry and Biophysics, University of California, ((S. Svetina, V. Heinrich, V. Kralj-Iglic, and B. Zeks)) San Francisco, CA 94143-0448. Institute of Biophysics and J. Stefan Institute, University of The actin cytoskeleton plays an important role in early C. elegans development. The Ljubljana, 1000 Ljubljana, Slovenia. experiments of Hill and Strome (1988, 1990) showed that disruption of filamentous (F-) actin in the one cell embryo results in a mis-positioning of both developmental Cell polarity is characterized by a nonuniform, directional molecules and the mitotic spindle. To study the role of the actin cytoskeleton during distribution of the constituents of the cell membrane and the early embryogenesis, we have isolated 17 potential actin binding proteins from C. cytoplasm. A physical mechanism for the occurrence of such a elegans oocytes using F-actin affinity chromatography. These C. elegansactin polar cellular organization is proposed, based on the notion column binding proteins (CABPs) bind tightly to columns containing F-actin, do not that the interaction of membrane embedded proteins with the bind control albumin columns, and are highly enriched relative to their abundance in surrounding membrane moiety must depend on membrane curvature. whole cell extract. We have raised mouse polyclonal sera against six CABPs. Using Because for any non-spherical cell the membrane curvature var- immunofluorescent confocal microscopy, four of these, CABP1, CABP14, CABP1 1, and CABP13, show localization to subsets of the actin cytoskeleton in fixed early ies over the cell surface, the energy of these proteins depends embryos. CABP1 staining co-localizes with actin in the cortex of early embryonic on their position on the membrane. For thermodynamic reasons it cells, but, unlike actin, is noticeably weaker at cell-cell boundaries. CABP14 staining is expected that the embedded proteins appear less frequently associates with the nucleus of cells in prophase, the entire cortex and cytoplasm of in membrane regions where the interaction energy is high, and cells in metaphase, but exclusively with the cleavage furrow of cells in telophase. accumulate where it is small. This concept is illustrated by a The most dramatic asymmetries are seen with CABP1 1 and CABP13 staining. theoretical analysis considering cells with prolate axisymmet- Unlike actin, which surrounds the entire cortex of the1 -cell embryo, CABP1 1 ric shapes. The cell shape and the lateral distribution of mob- transiently associates with the anterior cortex of the early 1-cll embryo. CABP1 3, ile membrane components are obtained by minimization of the on the other hand, associates asymmetrically with the lateral cortex (dorsal-ventral or membrane free energy. In the absence of proteins, this energy left-right) of 1-cell embryos, providing the first evidence that there is lateral is governed by the membrane elastic energy, and the resulting asymmetry this in C. elegans embryogenesis. CABP1 1 has been cloned and early shape of a closed, laterally homogeneous membrane is axisymmet- shows some homology to a known actin binding protein that is asymmetrically and has an additional equatorial mirror localized in Dictyostelium cells. Preliminary experiments in which CABP1 1 maternal ric symmetry. The in- clusion of the free energy of embedded function was eliminated using antisense RNA indicate that it an essential gene distributional molecules important for early development. Our data support the results of Hill and Strome may cause the cell to loose the equatorial mirror symmetry. In suggesting that theaction cytoskeleton is intimately involved with generating this case, the resulting distribution of membrane embedded mol- asymmetry in the 1-cell embryo. ecules over the asymmetrical shape becomes polar.

682 683 POLARIZED SECRETION OF FIBRINOGEN BY LUNG EPITHELIAL CELLSIN SUPPRESSORS OF UNC-73 DEFINE SUP-39, A NEW GENE INVOLVED IN CELL RESPONSE TO INFLAMMATION. ((G Guadiz, LA Spom, RA Goss, SO POLAR=TY IN C. ELEGANS ((Jin-Quan Run and Jeffrey C. Way)) Nelson Biol. Labs, Lawrence, VJ Marder and PJ Simpson Haidaris)) Dept. Medicine, Microbiology Rutgers University, Piscataway, NJ 08855 and Immunology, and Pathology, Univ. of Rochester, Rochester, NY 14642. In C. elegant, the unc-73 gene encodes a protein with a region homologous to yeast to The acute phase response (APR)is comprised of a series of immediate CDC24, a guanine-nucleotide-exchange factor for CDC42. Smilar CDC24, Unc-73 also reactions occurring at the onset of inflammaton. APR proteins, suchas plays a role in controlling cell polarity. Animals mutated in unc-73 are severely to axon also fibrinogen (FBG), act systemically in infection, wound repair, and the uncoordinated due incorrect guidance. We foundthat they exhibit defects in cell migration and asymetric cell division.The coordinated revertants ofunc-73(e936) we restoration of homeostasis. FBG gene transcription occurs in lung epithelial isolated include intragenic suppressors and extragenic dominant suppressors sup-39fjeS or cells only after with mediators resulting assembly stimulation proinflammatory in je6). sup-39 mutations alone have a wild-type movement as larvae and adults. However, and secretion of intact FBG. This study examines the polarity of FBG secretion they have the following novel and dominant phenotypes with incomplete penetrance: 1) bylung epithelium using A549 cells cultured on polycarbonate membrane Maternal embryonic lethality. The lethality decreases when the mother's age increases; 2) fiters. After IL-6 + dexamethasone induction and metabolic labeling, FBG Two instead of one row of oocytes in each gonadarm; 3) Left-handed Roller after L4 purified from apical and basolateral chambers was analyzed by SDS-PAGE. stage, which is much enhanced when synthetic with jell, a spontaneous mutation. These The results showed that newly-synthesized FBG was secreted from A549 cells results suggest that the sup-39 mutations probably have effects in many cell-polarity in a polarized manner, with 80 ± 5.1% of protein secreted basolaterally, events, especially in axon guidance, in choosing a single site on the gonad from which suggesting that FBG is secreted vectorially to the basement membrane in vivo. oocyte nuclei will emerge, and in the polar secretion of cuticle. However, sup-39 does not Immuno-electron microscopy confirmed these results. Depolymerizatlon of suppress the weak defect of asymmetric mec-3 expression inunc-73 mutant. sup-39 was micro-tubules withcolchiine not only altered the predominantly basolateral mapped between dpy-10 and cdc-42 and within 0.015 map units ofunc-104(11). Thus far, secretion, but the secretary pathway was blocked, resulting in intracellular no recessive alleles of sup-39 has been isolated,perhaps because of haplo-insufficiency of this gene (Run et.al, Genetics 143:225,1996). To further understand the role ofunc-73 and accumulation of FBG. As an APR protein, FBG functionsin bloodcotting, sup-39 in controlling cell polarity, we plan to clone sup-39 by using the fibrinolysis, and modulation of the immune response. In addition, FBG following strategies: 1) Get a more accurate position of by using TcI transposons in degradation products stimulate the of fibroblasts which produce sup-39(je5) proliferation Bergerac strains as markers; 2) Synthesize potentialsup-39 gene from mutant genome by matrix components and cytokines. Fibrin at the site of inflammatory formed PCR and then detect which will suppressunc-73; 3)Detect the position ofsup-39 mutation pneumocyte injury may as a matrix for epithelial and in function cell migration in potentialcoding sequences by examining single-strand polymorphism ofcDNA the maintenance of cellular polarity during cellswound repair. We show that the produced by unc-104 and its surrounding genes from N2 andsup-39 mutants; 4) Create a majority or FBG secreted from lung epithelial is directed toward the ECM, genomic library from sup-39(je5), findcosmids around unc-104, and then finda cosmid suggestive of a specific, localized APR under inflammatory conditions. which will suppress unc-73(e936). 118a Cell Polarity (684-688). Sunday

684 685 CLEAVAGE PLANE ORIENTATION AND CHOICE OF HANDEDNESS IN C. ELEGANS EMBRYOS. A GERMMLINE 14-3-3 ISOFORM IS REQUIRED FOR ASYMMETRIC ((Dominique Bergmann and William B. Wood)) Dept. of MCD Biology, University of Colorado, Boulder, CELL DIVISIONS ((Wenfu Wang', Daryl Dichoso', Kenneth J. Kemphues2, CO, 80309. and Diane C. Shakes 12,3.)) 'Dept. of Biology, University of Houston, Houston, TX; 2Section of Genetics and Developnient, Cornell University, Wild type C. elegant embryos, larvae and adults have a dextral handedness which is established by the skewing of the spindles of two blastomeres, ABa and ABp, during their L/R cleavage in the 4-6 cell Ithaca, NY; 3Dept. of Biology, College of William and Mary, Williamsburg, embryo (Wood, 1991). #143 (originally isolated and kindly provided by L. S. Rose, U.C. Davis) is a VA. maternal-effect mutation with defects in early cleavage plane orientations. Of particular interest to our Members of the highly conserved and ubiquitous 14-3-3 protein family ap- study, the #143 mutation also results in temperature sensitive lethality and handedness reversal. At pear to serve as mediators of protein-protein interactions, particularly in signal 16 C, #143 hermaphrodites produce viable progeny, 6% of which are sinistral. At 25° C, #143 transduction pathways. We have recently identified two 14-3-3 genes in the hermaphrodites produce 70% inviable embryos, and among the survivors, 40% are sinistral. Both the nematode C. elegans. ftt-1 (fourteen-three-three) (IV) encodes three tran- reversed adult handedness and the lethality can be traced back to defects in early cleavage plane scripts which are germline enhanced in their expression. ftt-l transcripts are orientations. In N2 animals, the skewing of the spindles in ABa and ABp places their left daughters abundant in hermaphrodite gonads and early embryos, but their levels de- slightly anterior to their right daughters. In a series of recordings made of embryos from #143 crease In encodes a hermaphrodites at 20- C, we observed a randomization of the orientation of the ABa and ABp spindles. during embryogenesis. contrast, ftt-2(X) single transcript which is on Occasionally ABa and ABp underwent a UR cleavage where the spindles skewed to place the right somatically expressed and rapidly turns in 2-4 cell embryos. ftt-1 daughters anterior (4/18 embryos), and this orientation leads to sinistral animals (Wood, 1991). ABa antisense RNA injections into hermaphrodite gonads yield defective embryos and ABp also occasionally divided ANP (2/18 embryos) or DN (2/18 embryos), or divided in different with symmetric rather asymmetric early cell divisions. These defects pheno- planes from each other. Embryos from hermaphrodites reared at 25° C exhibited earlier and more copy those of par-5 mutants, the only maternal effect lethal mutant identified severe cleavage defects, in agreement with L. S. Rose's initial observations. In temperature-shift in this genetic region. The six par genes help establish the anterior-posterior experiments, we have determined the temperature-sensitive period (TSP) of #143 for both the lethality embryonic axis in newly fertilized zygotes, a process which includes asym- and reversal phenotypes. The beginning of the TSP for both phenotypes is before fertilization. The end metric positioning of germline P-granules, a physically asymmetric first cell of the TSP for lethality is during the 4-cell stage, and the end of the TSP for reversal is between the division, and subsequent cell divisions which are distinct in both their tim- early 6-cell stage and the 8-cell stage. The TSP data is consistent with the hypothesis that the product ing and cleavage orientation (Kemphues et al., 1988; Shakes, Morton, and of the gene defined by #143 is required during the second AB division to establish proper embryonic Microfilament inhibitors cause handedness. Our immediate goals are to further characterize the cleavage plane defects, to look at Kemphues, unpublished). similar defects, sug- interactions between the gene defined by #143 and other genes involved in patterning the early gesting a connection between actin and the par proteins (Hill and Strome, embryo, and to clone the locus. 1990) Our conclusion that ftt-1 = par-5 is further supported by our obser- Reference: Wood (1991) Naiure 349:536-538 vation that FTT-1 protein levels are significantly decreased in certain par-5 mutants, and par-5 can be weakly rescued by ftt-1 containing cosmids. These studies suggest an important new role for 14-3-3 in mediating asymmetric cell divisions.

686 687 A GENETIC SCREEN FOR MUTATIONS THAT AFFECT NUCLEAR APPLIED ELECTRIC FIELDS INHIBIT POLAR AUXIN TRANSPORT. ((N.J. Holdaway, POLARITY AND ORIENTATION IN DROSOPHILA; EMBRYOS. ((H. N.A. Walker and R.L. Overall)) School of Biological Sciences, University of Sydney, Francis-Lang, N. Dompr, S. Lall' and W. Sullivan.)) Dept Biology, NSW 2006, Australia. (Spon. by J. Marc) University of California at Santa Cruz, CA 95064, USA and I ICRF, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. In plant tissue and cells, polarity is manifested by the polar transport of auxin (indol-3- acetic a In early Drosophila; development, nuclei are polar and maintain a strict acid; IAA), growth hormone involved in differentiation and tropic responses. orientation during the cortical syncytial divisions that take place at the embryo Regardless of orientation and against concentration gradients, auxin is transported basipetally, away from the tissue's apex. This preferential transport can be explained surface. Evidence for nuclear polarity, comes from structural studies (1) and by the asymmetric distribution of IAA-effluxors in the basipetal membrane1, as observations that gene may pair-rule segmentation transcripts be exported proposed in the chemiosmotic theory2. The mechanism by which putative IAA- directionally from such nuclei (2). Using the DNA binding dye daunomycin, effluxors may be localised to the basal membrane is unknown, however has been which specifically highlights the AT rich centromeric heterochromatin, it is suggested that endogenous electric fields may control the direction of auxin flow by apparent that nuclear orientation has already been established in nuclei prior electrophoresing these proteins within the plasma membrane3. We have tested this to their migration to the surface (S.L., HF-L and D. Ish-Horowicz). Due to hypothesis experimentally by monitoring IAA transport after application of overriding the nature of this process, we have performed a screen to identify temperature electric fields, both in the direction reinforcing and opposing the tissue's sensitive mutations in the large and relatively unexplored class of genes that endogenous polarity. Electric fields (10mV/cell;4h) were applied to 6 day old corn function both maternally and zygotically. Daunomycin staining is being used coleoptile segments by passing 0.4 mA of direct current through a weak salt solution to identify maternal effect mutants that specifically disrupt the polarity and in which the segments were suspended. Auxin transport was assessed by orientation of nuclei. We are currently using this technique to characterise measuring the distribution of a 'pulse' of radio-labelled IAA that had been applied to 170 X-linked ts lethals that we have isolated. 1) Hiroaka, Y., Agard, D.A. and one end of the tissue segment. The effect of electric field pretreatment on auxin Sedat, J.W. (1990) J. Cell Biol., 111, 2815-2828. 2) Francis-Lang, H.L., Davis transport was dependent on the polarity of the applied field with respect to the tissue's electrical When I. and Ish-Horowicz, D. (1996) EMBO J., 15, 640-649. endogenous polarity. the applied field polarity was the same as the endogenous (apex -ye), basipetal IAA transport was not significantly effected. However, when the applied field opposed the endogenous (apex +ve), transport velocity and total amount IAA transported decreased to 67±7% and 52±1 1% of the control respectively. Confirmation that these results are consistent with a redistribution of IAA-effluxors is currently being sought by use of an iterative computer program which models auxin transport characteristics. 1. Jacobs, M. and Gilbert, F.S. (1983) Science 220:1297-1300 2. Rubery, P.H. and Sheldrake, A.R.. (1974) Planta 118:101-121 3. Raven, J.A. (1979) New Phytol. 82:285-291

688

SIGNALLING SYSTEMS REGULATING POLLEN DEVELOPMENT IN NICOTIANA TABACUM ((Laura Zonia, Chris Staiger, Jaroslav Tupy.)) Biology Dept., Purdue Univ., W.Lafayette, IN 47907 Institute of Experimental Botany, Czech Academy of Sciences, Prague, CR Critical developmental transitions in higher plant cells are frequently pre- ceded by a polarization or asymmetrical reorganization of cellular components and/or organelles. There are two points in pollen development where a highly polarized cellular organization occurs. The first is during the asymmetrical microspore mitosis, resulting in a pollen grain containing the unequal vegeta- tive and generative cells. The second is during pollen germination, resulting in formation of a tip-growing extension of the vegetative cell called the pollen tube. Concurrently with these asymmetrical reorganizations, changes in cell shape and nuclear positioning are prominent developmental features. We have recently initiated a study to understand how cellular polarization during pollen development is established and maintained, and how the developmental pro- gram can be blocked or switched if cellular polarization is disrupted. Our initial work has focused on the actin cytoskeleton, inositol phospholipids, and calcium. We are also utilizing pharmacological studies to elucidate a func- tional role for these components during pollen development. We willpresent our most recent data from these studies. Sunday. Invertebrate Development (689-694) 119a

689 690 MECHANISM OF AN EVOLUTIONARY CHANGE IN MUSCLE CELL MYOGENIC DETERMINANTS IN THE ASCIDIAN EGG ((Janet DIFFERENTIATION IN ASCIDIANS WITH DIFFERENT MODES OF Chenevert and Christian Sardet.)) URA 671 CNRS/UPMC, Marine Cell DEVELOPMENT. ((T. Kusakabe,'23 B.J. Swalla,' N. Satoh,2 and W.R. Biology, Villefranche-sur-mer, France Jeffery1)) Marine Lab., University 1Bodega of California, Davis, Bodega Bay, The fertilized ascidian egg contains cytoplasmic domains of distinct cytoskele- CA 94923, 2Department of Zoology, Graduate School of Science, Kyoto tal and organelle composition in which determinants developmental University, Kyoto 606-01, Japan. 3Present address: Graduate School of of fate Hokkaido T. are localized. One of these domains, the myoplasm, contains molecules which Science, Univ., Sapporo 060, Japan. (Spon. by Kusakabe.) specify muscle development in the cells which inherit it after cleavage. We We have investigated the mechanism of an evolutionary change in ascidian wish to identify these muscle determinants and to elucidate their mechanism muscle cell development. The ascidians Mo/gula oculata and Mogula occulta of localization and segregation. We have undertaken two approaches. The are closely-related species with different modes of development. M. oculata first is to isolate ascidian homologs of the myogenic bHLH factors. Our first embryos develop into conventional tadpole larvae with a tail containing clone, "PMF", encodes a MyoD-like protein. It is expressed transiently dur- striated muscle cells, whereas M occwa embryos develop into modified ing embryonic development, starting prior to gastrulation at about the 64 cell tailless larvae lacking differentiated muscle cells. MocuMAl is a single copy, stage. Spatially, PMF expression is confined to the myoplasm-containing blas- larval-type muscle actin gene of M. oculata. MocuMA 1 mRNA first appears in tomeres, which are those which will give rise to the muscle cells of the the prospective muscle cells during gastrulation, and transcripts continue to tadpole tail. Analysis of the in vivo function of PMF is underway. Since PMF message be present throughout embryogenesis. Muscle adtin mRNA was not detected dunng M. occulta embryogenesis. Interspecific hybrids produced by fertilizing is absent in the egg, it is unlikely to be the primary determinant in the my- M. occuta eggs with M ociata sperm recover the ability to express muscle oplasm. We are therefore searching for other factors, of the MyoD and MEF2 actin mRNA in vestigial muscle cells, suggesting that trans-acting factors families, which could function upstream of PMF. An alternative approach to responsible for muscle actin gene expression are conserved in M. occLta. identify determinants relies on function rather than homology. Egg messages The presence of these trans-acting factors was confirmed by showing that are microinjected into nonmuscle blastomeres and are screened for their abil- the MocuMAl/lacZfusion construct is expressed in the vestigial muscle cells ity to promote a myogenic fate in the embryo. Positive clones are amplified of M. occulta larvae. The orthologous larval muscle actin genes MoccMA la and purified. Our model is that the myoplasm-localized determinant, of as yet and MoccMA lb were isolated from an M. occilfta genomic library. The coding unknown identity, acts to turn on PMF in the cells which inherit it. Future regions of these genes contain deletions, insertions, and codon substitutions work will address the mechanism of this that would make their products non-functional. Expression of MoccMA la/lacZ activation. and MoccMA lb/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMA lb. These results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression.

691 692 LOCALIZATION AND MOVEMENT OF CYTOPLASMIC DOMAINS IN DYNAMICS OF MITOCHONDRIAL DISTRIBUTION IN EARLY C. ELEGANS EMBRYOS ASCIDIAN ZYGOTES ((Fabrice Roegiers and Christian Sardet.)) URA 671 ((Ananthkumar S. Badrinath, John G. White)) Laboratory of Molecular CNRS/UPMC Station Zoologique, 06230 Villefranche sur Mer, 06230, France Biology, University of Wisconsin, Madison Madison, WI 53706 In the egg of the ascidian Phallusia mammillata, fertilization triggers the formation and reorganization of distinct cytoplasmic domains in two phases. Mitochondria were labelled with a mitochondria-specific probe, Rhodamine A first phase occurs shortly after fertilization and is triggered by the activating 6G (R6G), by growing worms on 2.5 ug/ml R6G-NGM (nematode growth medium) Ca++ wave (Roegiers et al, Development 121, 1995), the second occurs after plates. Embryos at pronuclear migration stage were dissected out of the worms the completion of meiosis and depends on the displacement and growth of the and their mitochondria were visualized using a confocal microscope. Image analysis large sperm aster. Two main domains are relocalized, the mitochondria-rich was performed using NIH Image software. Following the first cell division, the myoplasm, and a domain of Endoplamic Reticulum (ER) accumulation that mean fluorescence intensity in the posterior blastomere, P1, was found to be forms during the first phase. The myoplasm, labelled with DiO(C2)3, ER approximately 20 percent greater than in the anterior blastomere, AB. Preceding with DiICse(3), and microtubules labelled with Rhodamine, were observed in this division, during a stage known as 'pseudocleavage', mitochondria deep within vivo using a confocal microscope. During the second phase of relocalization, the embryo flowed posteriorly toward the sperm pronucleus, and mitochondria both the ER domain and the myoplasm migrate into the center of the sperm located close to the cortex flowed in the opposite direction. In contrast to these aster and with it away from the membrane surface. This movement depends flows that take place in the posterior half of the embryo, there does not appear to on the presence of microtubules. Before this bulk movement and immediately be any directed flow of mitchondria in the anterior half either deep within the following meiosis, the second phase begins with a transient protrusion ("veg- embryo or close to the cortex. The rate of migration of mitochondria deep within etal button"). The vegetal cortex then begins to vibrate. This is followed by the embryo was 11.84 +/- 2.37 um/min (n=14, 3 embryos), and the rate of a surface contraction wave just prior to the onset of mitosis. None of these migration of the cortical mitochondria that were flowing in the opposite direction surface movements appear to be microtubule dependent, as all of the events was 14.09 +/- 3.71 um/min (n=19, 3 embryos). The ratio of mean intensity in the occur in the presence of 1.2 uM nocodazole. In treated eggs the sperm aster posterior cortex to the anterior cortex appeared to increase linearly during does not form, the second phase of ooplasmic segregation is incomplete, and pseudocleavage. The flows apparently enriched the posterior cortex and depleted the eggs fail to undergo mitosis. the anterior cortex of mitochondria. We optically sectioned through a two-cell embryo, and found that the density of mitochondria in the P1 cortex was clearly greater than in the AB cortex. We suspect that there is a mechanism involved in trapping' mitochondria to the posterior cortex during pseudocleavage, and that this could possibly explain how the difference in mean fluorescence intensity between P, and AB blastomeres is established.

693 694 CRYSTAL PROTEIN EXPRESSION DURING VEGETATIVE DIFFERENCES IN DNA LEVELS DURING EARLY CLEAVAGE GROWTH OF DICTYOSTELIUM ((Kristy K. Taylor and Catherine P. STAGES OF MESOCYCLOPS EDAX AND MESOCYCLOPS Chia)) School of Biological Sciences, University of Nebraska-Lincoln, NE. LONGISETUS (CRUSTACEA, COPEPODA). ((E.M. Rasch and G.A. Wyngaard)). East Tennessee State University, Johnson City, TN We are studying interactions between membrane proteins and the 37614 and James Madison University, Harrisonburg, VA 22807. cytoskeleton of Dictyostelfium discoideum. Detergent (Triton X-100) Presumptive somatic cells become different from primordial germ -insoluble cytoskeletons from vegetative (AX2) amebae are enriched in a 70 kDa Concanavalin A (Con A)-binding protein (gp7O). An enriched fraction cells in several species of copepods by the process of "chromatin diminution' (Hacker, V., Arch. mikr. Anat. 48: 759, 1894) duringwhich of gp7O was prepared by Con A affinity chromatography of cytoskeletons certain chromosome segments are excised and eliminated at an early from axenically-grown cells. N-terminal amino acid sequence of gel-purified embryonic mitosis from putative somatic cells but are retained in germ gp7O established its identity to a69 kDa 'crystal protein' previously line cells. To monitor DNA content during embryogenesis, Feulgen- characterized by Bomblies, et al. (1990, J.Cell Biol. 110:669 -679). Enzymatic stained adult male and female somatic tissues and nuclei from 4-16 deglycosylation of gp7O resulted in a65 kDa product that did not bind Con cell embryos were measured with a Vickers scanning cytophotometer, A, verifying the predicted number of N-glycosylation sites. Bomblies, etal. using chicken blood cells stained simultaneously with each series of showed crystal protein (gp7O) to be developmentally regulated, and with its copepod slides as an internal reference standard of 2.5 pg DNA/cell. homology to esterases, suggested to be involved in spore case degradation. The 2C DNA level for adults of M. edax is 3.0 pg DNA, whereas, the We find crystal protein expression to be influenced by growth conditions. As 2C DNA level for M.Iongisetus is roughly 1.8 pg DNA. The large, determined by immunoblotting, axenically-grown, vegetative cells express densely stained germ line nuclei of M. edax contain 32-36 pg DNA/ moderate levels of crystal protein. When bacteria are the sole food source for nucleus in 4-8 cell embryos, which clearly show separation of maternal both wild type and over-expressor cell lines grown in suspension, crystal and paternal chromosomes (gonomery) at both metaphase and in these protein levels are reduced significantly in vegetative cells. However, in these anaphase early cleavage stages. Measurements of mitotic configurations during the 4th cleavage (chromatin diminution) show same cell lines grown on bacteria, crystal protein is expressed late in that 75% of the total DNA remains at the equator during late anaphase development. The differences in crystal protein expression may indicate that in M. edax, but only 35-38% of the total DNA (11-12 pg) accumulates it is regulated during vegetative growth and during development. at the equator in M. Iongisetus. The biological significance and function of chromatin diminution remain unknown. 120a Invertebrate Development (695-700). Sunday 695 696 GLYCOGEN SYNTHASEACTIVITY IN DEVELOPING ARTEMIA. CALMODULIN POINT MUTATIONS EFFECT DROSOPHILA ((E. Yi B.S. Khatra, and R.A. Acey)) Department of Biological Sciences DEVELOPMENT AND BEHAVIOR ((H.B. Nelson, R. Heiman, C. and the Department of Chemistry and Biochemistry, California State Bolduc and K. Beckingham)) Department of Biochemistry and Cell University, Long Beach, CA 90840 Biology, Rice University, 6100 Main Street, Houston, TX, 77005-1892.

The brine shrimpArtemia has proved to be a unique biological system for Eight Drosophila mutations have been isolated that produce death or studying the biochemical and genetic mechanisms controlling early reduced viability in combination with a null allele of the calmodulin (Cam) embryonic development We report here on the developmentally regulated gene. Six of these mutations produce single amino acid changes to the expression of glycogen synthase in Artnemia. Embryos were collected, Cam protein and include mutations in residues that are known to homogenized and assayed for glycogen synthase activity by incubation of coordinate calcium or interact with particular targets. Various the extracts with glycogen and UDP-14C-glucose. Enzyme activity is combinations of these mutations produce adult morphological defects expressed as the amount of radioactivity incorporated into ethanol such as ectopic wing veins or melanotic scabs. One mutation is pupal precipitable material/mg protein. Glycogen synthase activity remains low lethal in the hemizygous condition and individuals die with a striking during the first 12 hours of development, steadily increases to its maximum developmental defect the adulthead fails to evert and develops inside the level during the next 12 hours of development and then returns to the initial thorax. Maternal effects on embryogenesis are also seen with some low level of activity within 12 hours. Concomitantly, the embryonic level of mutant combinations, the most distinctive being failure of the glycogen peaks at 24 hours of development (231 protein) and then chromosomes to separate during anaphase. Finally, several Cam mutants Rg/mg show behavioral defects, such as poor coordination and flightlessness. declines to 57 ag/mg protein. The glycogen synthase activity is glucose-6- These mutants demonstrate a role for Cam in a number of developmental phosphate dependent, suggesting that the observed levels of activity are due and neuronal processes. to differences in the cellular level of the enzyme. Glycogen synthase activity is associated with glycogen and can be solubilized by treatment of the polysaccharide with salivary amylase. Treatmentof the solubilized enzyme with phosphatase increases its activity ratio from 0.027 to 0.25, suggesting that the enzyme activity may also be regulated by phosphorylation. Supported by MBRS Grant GM08238

697 698 CATALYTIC PROPERTIES OF PROTEASOMES FROM ECDYSIS STUDIED BY THE USE OF A TEMPERATURE SENSrTIVE DEVELOPMENTAL MUTANTS OF DROSOPHILA ((D.L. Mykles, J. MUTANT (shibire"') IN DROSOPHILA MELANOGASTER. ((K. Ikeda and Covi, and J.M. Belote.)) Biology, Colorado State University, Ft. Collins, CO J.H. Koenig)) Division of Neurosciences, Beckman Research Institute of the City 80523 and Biology, Syracuse University, Syracuse, NY 13244 of Hope, Duarte, CA 91010. The proteasome is a large (20 S) multicatalytic proteinase complex that cat- alyzes ATP/ubiquitin-dependent proteolysis in eukarotes. To determine the The neuromuscular system involved in ecdysis, by which a pupa emerges from its role of proteasome during development, we have analyzed biochemical prop- erties of wild-type (WT) and dominant temperature sensitive (DTS) mutant puparium, offers a useful material for developmental studies. In the temperature proteasomes in Drosophila. For DTS-5 and -7 mutants, heterozygous individ- sensitive mutant, shibire"' (shi), endocytosis is normal at 191C (permissive uals raised at restrictive temperature (29 C) die as late pupae, with reduced temperature) but it is reversibly blocked at 290C (restrictive temperature). With imaginal disk derivatives and missing adult abdominal structures. At 25 C, this mutant, if a restrictive temperature pulse (295C, 3 hrs) is applied during its heterozygotes have normal viability and look normal. However, at 25 C, ho- pupal stage, the temperature effect appears only in particular muscles. It was not survive to adulthood. The DTS5 gene encodes a proteasome mozygotes do found that every muscle has its particular timing forthe initial division of the beta subunit [Saville and Belote (1993) PNAS 90:8842-8846)]. While DTS7 primordial myocyte, as well as for syncytium formation, which occurs later, and has yet to be cloned, genetic evidence strongly suggests that it is function- that these are the two critical times when muscle development ally related to DTS5. Proteasomes were purified from adult WT, DTS5/+, is affected by the and DTS7/+ flies raised at permissive temperature; chromatographic separa- temperature pulse. Ecdysis is performed by particular sets of neuromuscular tions on arginine-Sepharose, Mono Q anion exchange, and TSK-phenyl-5PW systems which develop, function and degenerate only for this purpose in a short columns were similar between the three preparations. Trypsin-like activity of period of time (2 days to develop, 5 15 min to function and 12 hrs to degenerate). DTS5 and DTS7 proteasomes was lower than that of WT enzyme, while neu- One of these muscles, the transverse ptilinum muscle (rpm) starts to develop at 68 activity was greater in DTS7 proteasomes than that in tral chymotrypsin-like hrs from prepupa formation When the temperature pulse is applied at this WT and DTS5 proteasomes. In contrast, peptidylglutamyl-peptide hydrolase (19QC). stage, a fly missing this muscle is created. Apparently the division of the activity was the same in all three preparations, and was stimulated to the myocyte same extent with low concentrations of SDS. DTS mutations apparently alter primordial is blocked. If the temperature pulse is applied at the 76 hr the catalytic properties of the proteasome, which result in enzyme disfunction stage, a fly having a giant tpm is created. This giant tpm is functional, so that the at elevated temperatures. Supported by NSF. fly can break the puparium to expose its head. The function of the tpm is to keep the internal pressure for the extruded ptilinum. Immediately after ecdysis, the tpm stms degeneration. After 12 hrs it disappears completely and the space is replaced by an air sac.

699 700 CHARACTERIZATION OFTHE HOMEOTICTARGET GENE EPIDEPM4L STRIPES THE EXPRESSION OF A MOLT PROTEIN DURING AND PATCHES, A DROSOPHILA HOMOLOGUE OFTHE HUMAN DIASTROPHIC DEVELOPMENT OF AN INSECT, CALPODES ETHLIUS DYSPLASIA GENE. ((N. Roche. T. Kaufman)) Dept. of Biology and Howard ((O. Marcu, M. Locke, B.G. Atkinson)) Department of Zoology, Hughes Medical Institute, Indiana University, Jordan Hall, Bloomington, IN University of Western Ontario, London, Ontario, Canada NBA 557. 47405. A protein associated with molting has been isolated from larval Epidermal stripes and patches (easp) was isolated through an cuticles of the butterfly Calnodes ethlius at the time of pupation. immunopurification technique in an effort to identify Antennapedia target Antibody reactivity showed the protein in hemolymph of the larval genes. Modulation of esp expression by the homeotics occurs negatively by intermoit when the mRNA is expressed in epidermis and fat body, Antennapedia and positively by Ultrabithorax, abdominal A, and Sex combs but the protein only appeared in cuticle and fat body prior to moting. reduced. Esp shows high levels of similarity to the human gene for The protein is probably stored in the hemolymph throughout the Diastrophic dysplasia (DTD) which encodes a novel sulfate transporter intermolt until it is transported to the cuticle and molting fluid. This (Hastbacka, J., Cell 78, 1994 (6): 1073-1087). DTD patients show tissue distribution shows how secretion at an earlier stage in impaired proteoglycan synthesis and cartilage defects that result in development provides an abundant source of protein that is only dwarfism(Hastbacka, 1994). To understand the role that esp plays in the mobilized later for transport to the cuticle, prior to ecdysis. A cDNA development of the fly and in the process of segment identification, ectopic encoding the protein was isolated by antibody screening of an expression experiments with both sense and antisense esp are being done. Ai epidermal library. The aminoacid sequence derived from the cDNA EMS mutagenesis screen for F2 lethals using a deficiency that removed easp contains a hydrophobic signal peptide on its N-terminal end was performed and candidate asp mutant alleles have been identified. Esp followed by a sequence showing 51% similarity with an amidase expression data supporting the modulation of Esp by homeotics will be show which cleaves the bond between N-acetylmuraminic acid and L- and progress towards the identification of an esp allele and an asp phenotype alanine. This enzyme would be a first step in degrading the chitin- will also be presented. peptide bonds in old cuticle at molting. The protein was found in all larval instars and pupae but was absent in adult insects, as would be expected if it was involved in digestion of larval and pupal cuticles at the time of larval-larval, larval-pupal and pupal-adult transformation. Sunday. Invertebrate Development (701-706) 121a 701 702 SUBUNIT ASSOCIATION AND LIGANDS FOR SEA URCHIN SEA URCHIN MEF2 IS EXPRESSED DURING EARLY INTEGRINS. ((P.L. Hertzler and D.R. McClay)) Department of Zoology GASTRULATION ((J.M.Venuti, R.L. Beach* and N.S. Gentile.)) & DCMB Group, Duke University, NC 27708. Department of Anatomy & Cell Biology, College of Physicians & Surgeons of Columbia University, New York, NY 10032 and *Biology Department, We have isolated both a and integrin subunits from the sea urchin Hollins College, Roanoke, VA 24020 Lytechinus variegatus. The a subunit is related to the a5/aV/ac8/aIlb SUM-1, the sea urchin homolog of MyoD, is expressed concomitant with group of vertebrate integrins, while the subunit is related to the differentiation of muscle precursor cells cultured in vitro. This is prior to 131/32/137 group; neither sea urchin integrin has a specific vertebrate overt myogenic differentiation in the intact embryo, suggesting that SUM-1 is an early marker and potential activator of homolog. The a integrin RNA is expressed as an 11 kb transcript in myogenic commitment. Micro- injection experiments over-expressing exogenous SUM-1 in intact embryonic stages from egg through pluteus larvae. The p integrin RNA embryos show transactivation of co-injected muscle-specific reporters only in a is present as a matemal transcript of 10.5 kb which is degraded after sub- set of mesenchyme cells. This suggests SUM-1 activity is restricted either by fertilization. Expression of zygotic integrin RNA begins at the blastula cofactors present in mesenchyme or negative regulators in other lineages. To stage. association of these integrin subunits, we To study the potential understand how SUM-1 activity is regulated in the different lineages of the em- have produced polyclonal antibodies and performed bryo we have begun to search for other factors that interact with SUM-1. To immunoprecipitation experiments using sea urchin embryo extracts. assess whether sea urchin MEF2s play a role in restricting SUM-1 activity, we To study the ligand binding of these integrins, we have transformed have cloned and characterized a sea urchin MEF2 homolog. MEF2 (myocyte line B2 CHO cells, which express low levels of a5 integrin (Schreiner et enhancer factor-2) transcription factors bind to cis-elements in muscle-specific al., 1989, J. Cell Biol. 109:3157) with the sea urchin a integrin. genes that are often juxtaposed to the binding sites for the MyoD factors Immunoblotting with sea urchin a integrin antibody was performed to and may directly interact with these bHLHs. We screened a Lytechinus var- study the expression of sea urchin a integrin in transformed cells. iegatus late gastrula stage cDNA library using the MADS/MEF2 domain of Immunoprecipitation with mammalian 1 integrin antibody was Drosophila MEF2 as a probe. We identified two cDNAs whose deduced amino acid sequences show >85% identity to other MEF2s within performed to study the ability of exogenous sea urchin a integrin to the MADS/MEF2 domain. Northern analysis identified a single associate with endogenous 11 integrin. Transformed cells were plated transcript of approximately 7 Kb expressed initially in the early gastrula. RNase con- on various extracellular matrix substrates to determine specificity of protection analyses firm that sea urchin MEF2 is expressed during early gastrulation and precedes ligand binding. Cell adhesion assays were performed to determine the SUM-1 expression. strength of binding to various substrates. RNA injection experiments are in progress to study the role of integrins in sea urchin development.

703 704 EXPRESSION OF ENDODERM SPECIFIC GENES ARE EFFECTED DIFFERENTLY METALLOPROTEINASE INHIBITORS BLOCK SPICULE FORMATION BY BY INHIBITORS OF TWO DIFFERENT EXTRACELLULAR MATRIX PRIMARY MESENCHYME CELLS IN THE SEA URCHIN EMBRYO. MOLECULES((N.H. Luke and B.T. Livingston)) School of Biological Sciences, University ((E. P. Ingersoll and F. H. Wilt)) Department of Molecular and Cell of Missouri, Kansas City, MO 64110.) Biology, University of California, Berkeley, CA 94720. Expression ofS. purpruatus forkhead (Spfkh) peaks in the mesenchyme blastula then In order to determine the role of decreases in the gastrula and is limited to cells that undergo invagination in the early and matrix metalloproteinases mid gastrula. Endo 16 is first expressed in the vegetal plate of the hatched blastula and (MMPs) in sea urchin development, we treated sea urchin expression continues in the endoderm of the embryo through later stages. To determine if embryos with a variety of specific MMP inhibitors and either invagination or the extracellular matrix influences Sgfkh and endo 16 expression, determined the effects on morphogenesis. Synthesis of the gastrulation was inhibited and extracellular matrix processing was altered by exposing spicules of the larval skeleton by the primary mesenchyme cells embryos to 13-aminoproprionitrile fumerate (BAPN) and culturing embryos in sulfate free (PMCs) was dramatically and specifically inhibited by the MMP sea water (SFSW). Embryos cultured in SFSW do not invaginate and are arrested at the inhibitors TIMP2, BB-94 and ISN-3825. In all cases spicule mesenchyme blastula stage of development. Levels of Endo 16 were found to be lower in formation was initiated, but elongation was inhibited. SFSW embryos as compared to controls. After transferring the embryos to artificial sea Application of these inhibitors during spicule elongation water the embryos invaginated with-in forty five minutes; however, upon recovery, levels resulted in a rapid block in further elongation of the spicules. of Endo 16 were still lower than that of control embryos. fkch expression in embryos Expression of proteins present in the organic matrix of the cultured in SFSW on the other hand was elevated as compared to control embryos. calcerous spicule was not altered. However, the mRNAs encoding BAPN inhibits collagen deposition by repressing the collagen cross linking enzyme these proteins were present at about twice the levels in control lysyl oxidase resulting in arrest at the mesenchyme blastula stage. Although embryos. Analysis of polysome gradients indicates that all of morphologically the BAPN treated embryos were delayed relative to control embryos, these messages are loaded onto polysomes and should be they expressed Endo 16 to a greater extent then control embryos. In later stages levels of translated, suggesting that the levels of are Endo 16 in both control and BAPN cultures are nearly the same. BAPN cultures express these proteins regulated at some posttranslational level. Slfkh to the same extent as control cultures. These results suggest that disruption of the Inhibition of spicule apical extracellular matrix influences the expression patters of endol6 and Spfkh whereas formation is not due to blocking cell-cell fusion by PMCs since PMC fusion occurs collagen does not appear to influence expression of either gene. normally in MMP inhibitor-treated embryos. We are currently attempting to identify the enzymes that are inhibited by these MMP inhibitors and to determine the roles that they play in spicule formation.

71.5 706 MORPHOLOGIC AND BIOCHEMICAL RESPONSE OF SEA URCHIN EMBRYOS TO DIFFERENTIAL EXPRESSION OF ACTIN GENES DURING AMPHIOXUS METHOXYCHLOR. ((J.M. Mwatibo and J.D. Green)) Department of DEVELOPMENT. ((R. Kusakabel, T. Kusakabe2, N. Satoh', N.D. Holland3 Anatomy, Louisiana State University School of Medicine, New and L.Z. Holland3)) 'Division of Biological Sciences, Graduate School of Orleans, LA 70112. Science, Kyoto University, Kyoto 606-01, Japan, 2Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Developmental morphology and protein synthesis of sea urchin Japan, 3Scripps Institution of Oceanography, University of California San zygotes and embryos in response to an environmental toxicant, Diego, La Jolla, California, 92093-0202. (Spon. by N. Suzuki.) the pesticide methoxychlor (MXC), were investigated. Acute an exposure to MXC during the pre-hatching stages of Lytechinus The direct ancestor of vertebrates is thought to be animal that resembles present amphioxus. However, how various genes and their functions link the victus not only reduced the percentage of embryos undergoing developmental events in amphioxus and in vertebrates normal cleavage, but also altered the morphology of later still remains unclear. To investigate the relationships between the actin gene evolution and their stages. Gut development was stunted as was later development roles in development, we isolated a muscle and a cytoplasmic actin cDNAs of spicules. However, when hatched blastulae were exposed to from two closely-related species of amphioxus. Amphioxus was found to MXC no significant abnormalities were observed at later stages. have a vertebrate-type muscle actin gene, and its characteristic deduced A commercial monoclonal antibody to heat shock protein (anti- amino acid sequence implies that it diverged early in evolution. Transcripts of HSP 70) was used as a probe on immunoblots to ascertain this muscle actin gene appears in the somites and the myotomal expression synthesis of this stress protein. Western blots of homogenized continues throughout larval development. The muscle actin gene is also unfertilized eggs were not immunoreactive, while early cleavage expressed in smooth muscle cells associated with gill slits. Cytoplasmic actin stage embryos had constitutive HSP 70(HSP 73) immunoreactivity. gene expression begins in neural ectoderm and large part of mesoendoderm, to MXC exposure of hatched blastulae resulted in the detection of and becomes restricted the neural plate, notochord and pharyngeal tissue as development proceeds. In the neural plate, the mRNA was accumulated at the stress-inducible HSP 70 (HSP 72). A similar response was the base of cells where the neural plate contacts with the notochord. The observed when plutei were exposed to MXC. In MXC conclusion, 3'UTR of this gene contains a sequence that might correspond to "zipcode" exposure before hatching resulted in abnormal morphology in sequence of chick 0-cytoplasmic actin gene which is thought to direct embryos not synthesizing stress-induced HSP 72, while embryos intracellular mRNA localization. Further analysis of developmental mechanisms exposed after hatching synthesized HSP 72 and were normal. involving multiple actin genes in amphioxus is expected to provide a new insight into the evolution of chordates. 122a Sperm and Spermatogenesis (707-712). Sunday

707 708 THE LOCALIZATION OF MULTIPLE CATHEPSIN mRNAs IN THE IDENTIFICATION, MOLECULAR CLONING AND CHARACTERIZATION OF SEMINIFEROUS EPITHELIUM BY IN SITU HYBRIDIZATION IS CONSISTENT SPERM TAIL AUTO-ANTIGEN SA-ODF75. ((M.S.Dubova-Mihailova, K.L.Klotz, WITH THEIR ROLE IN GERM CELL(GC) MIGRATION. ((Sanny S.W. Chung'l2.Li-ji M.L.Baran, C.J.Flickinger, J.C.Herr)) Dept. of Cell Biology, University of Virginia Zhu', Meng-yun Mol, Will M. Lee2 and C. Yan Cheng'.)) 'The Population Council, 1230 York Avenue, New York, New York 10021; and 2Department of Zoology, The University of Health Sciences Center, Charlottesville, VA 22908 Hong Kong, HongKong, Hong Kong. Proteases play a crucial role in GC migration throughout spermatogenesis. Previous studies Rat hyper-immune serum raised against rat epididymal spermatozoa was used by RT-PCR have shown that Sertoli as well as Leydig cells express the mRNAs of cathepsin to identify and clone a highly conserved sperm antigen (SA-ODF75) by B, C, D, H, Land S while GC isolated from adult rat testes express all of the above cathepsins screening a human testis cDNA library. The major mRNA transcript for SA- except cathepsin D. However, GC, in particular spermatocytes and spermatogonia, isolated encodes a 75 kD from immature rat testes at 10-20 days of age also expressed cathepsin D. These results ODF75 protein. High stringency Northern blot analysis of mRNA from 16 human and 22 suggest an active involvement of GC in their migration. To examine if GC indeed express baboon tissues reveals testis specificity of multiple cathepsin mRNAs, we have localized the mRNAs of cathepsin D, H, L, and S in the expression of SA-ODF75. In Western blots of human, mouse and rat sperm epithelium at different stages of the cycle by in situ hybridization. Fragments of cDNAs extracts rabbit poly-clonal serum raised against human recombinant SA-ODF75 coding for cathepsins were prepared by RT-PCR, subcloned into pGEMR-T vector (Promega), identifies a number of protein bands with Mr 40-43, 53-55, 63-66, 73-75, 85-87 and their authenticity confirmed by direct sequencing. These plasmids were used as the kD. of these immunoreactive templates to synthesize the sense and anti-sense RNA probes which were labeled by random- Most proteins exist in multiple iso-forms primed incorporation of digoxigenin(DIG)-labeled dUTP. Frozen sections of testes were detectable on 2D blots. Comparative immuno-staining of 1D and 2D Western hybridized with DIG-labeled RNA probes. Thereafter, the sections were incubated with a blots of human sperm extracts with rabbit anti-SA-ODF75 anti-serum, rat hyper- sheep anti-DIG antibody conjugated to alkaline-phosphatase. The antibody bound DIG probe- immune anti-rat sperm serum or rat post-vasectomy serum prove that SA- mRNA duplexes were visualized by an enzymatic reaction. It was found that the sense probes ODF75 is one of the main human sperm proteins identified by rat post- did not yield any positive staining. Using the anti-sense probes, cathepsin L mRNA was found and to localize almost exclusively at the basal layer within the cytoplasm of Sertoli cells and vasectomy hyper-immune sera, suggesting that SA-ODF75 is a major spermatogonia in the epithelium. At stages VI-VII of the cycle just prior to the release of highly conserved sperm auto-antigen. Indirect immuno-fluorescence with elongate spermatids into the lumen, the signal of cathepsin L mRNA was so intense that it affinity purified rabbit anti-SA-ODF75 antibodies localize SA-ODF75 to the formed a complete dark stained precipitate at the basal lamina in the epithelium that encircled middle piece and proximal principal piece of the tail, with no detectable staining the entire tubule. At the beginning of stage VIII just prior to spermiation, no staining of of the distal end of the principal piece. Immuno-electron studies localize SA- cathepsin L mRNA was seen in the epithelium. For cathepsins D and S. their mRNAs were to the cortical area of found abundantly in the cytoplasm of Sertoli cells and spermatocytes in almost all stages but ODF75 outer dense fibers and their associated peaked at stages VII and VIII of the cycle. Very few cathepsin H mRNA was found in the structures. Sequence and predicted secondary structure similarity of SA- epithelium but largely restricted to the interstitium. Conclusion: (i) the expression of multiple ODF75 C-terminal domain to non-muscle myosin heavy chain tail suggests that cathepsin mRNAs in the testis is stage specific; and (ii) GC express multiple cathepsin it may be a member of the myosin family. (Supported by NIH HD29099) mRNAs suggesting they may play a more active role than anticipated in their migration.

709 710 CENTRIN EXPRESSION AND LOCALIZATION IN THE DEVELOPING A QUANTITATIVE STUDY OF THE PROPORTION OF POLYSOMAL MOUSE TESTIS DURING SPERMATOGENESIS. ((J.N. Glantz and J.L. mRNA AND RIBOSOME-SPACING OF 16 mRNAs IN MOUSE TESTIS. Salisbury)) Tumor Biology Program, Mayo Foundation, Rochester, MN 55905. ((L.M. Cataldo, M.A. Mastrangelo, and K.C. Kleene)) Department of Biology, University of Massachusetts Boston, Boston MA 02125. The Ca2+-binding protein centrin is a component of centrosomes, mitotic spindle poles, centrioles, and the basal bodies of cells with motile cilia or flagella. Previous studies have shown that centrin is essential for spindle pole The vast majority of mRNAs in meiotic and haploid spermatogenic cells exhibit separation in budding yeast and for flagellar excision in the green alga high levels of translationally inactive free-mRNPs, indicative of a block to the Chlamydomonas. During spermatogenesis, specialized microtubule-based initiation of translation. To investigate the mechanism of this inhibition, structures play essential roles in forming the meiotic spindle, reshaping the seminiferous tubules from adult mice were cultured briefly, and the distributions nucleus, and forming the sperm flagellar axoneme. We have used RT-PCR, of 16 mRNAs in various testicular were and immunohistochemical to elucidate the expressed cell-types analyzed by Western blotting techniques sedimentation of extracts on sucrose and measurement of association of two mouse centrin isoforms, mCenl and mCen2, with specialized cytoplasmic gradients microtubule-based structures of spennatogenic cells. Using oligonucleotide the levels of each mRNA in gradient fractions by densitometry and primers specific for either mCenl or mCen2 cDNAs, we have shown that mCen2 phosphoimaging of northern and slot blots. The results reveal that the proportions is constitutively expressed in a variety of mouse tissues, while mCenl expression of various mRNAs sedimenting with polysomes range from less than 15% to is limited specifically to developing testicular cells. Semi-quantitative RT-PCR greater than 80%, demonstrating that mRNA-specific mechanisms regulate the analysis reveals that the expression of mCenl mRNA increases dramatically initiation of translation. Most mRNAs in meiotic and early haploid spermatogenic during spermatogenesis coincident with the onset of meiosis in male germ cells cells and Sertoli cells are translated on in which ribosomes are on post-partum day 14. Immunoprecipitation and Western blot analysis of adult polysomes spaced and neonatal testis lysates using anti-centrin antibodies demonstrates differential about 80-100 bases apart on the coding region, the typical spacing in somatic expression of mCenl and mCen2 at the protein level. In mature spermatozoa, mammalian cells. However, the ribosome spacing on protamine 1 and 2 mRNAs immunostaining with anti-centrin antibodies labeled both the basal body and is much closer, 30-40 bases, demonstrating that in late haploid cells the rate of proximal centriole. Phosphospecific centrin antibodies label phosphorylated translational initiation is unusually rapid relative to the rate of elongation. centrin along a larger caudal portion at the basal plate of mature sperm nuclei. These results show that centrin is associated with the microtubule based Slowing translational elongation with cycloheximide has negligible effects on the structures of mature sperm. These studies also suggest that mCenl plays a proportions and ribosome-spacing of polysomal mRNAs in meiotic and haploid unique functional role in testis development through its specific expression in cells, indicating that competition for initiation factor eIF-4E, a major form of meiotic and post-meiotic sperm cells while mCen2 maintains a more-or-less translational control in somatic cells, does not account for the high levels ofthese constant level of expression in somatic cells. mRNAs in free-mRNPs.

711 712 ISOLATION OFTHE RAT SPERMATID MANCHETTE: BIOCHEMICAL AND STRUCTURAL CHAR- ALKALINIZING AGENTS INCREASE THE RESPONSE OF THE HUMAN ACTERIZATION. ((K. Mochida, L.L.Tres, and A.L.Kierszenbaum)) Departmentof Cell Biology and SPERM ACROSOME TO THE AGONIST, PROGESTERONE. ((Nicholas L. Anatomical Sciences, CUNY Medical School, New York, NY 10031. Cross)) Department of Anatomy, Pathology, and Pharmacology, Oklahoma State University, Stillwater OK. We have developed a procedure for the isolation of intact manchettes from rat spermatids of incubation in human spermiogenic steps 8-14.The procedure consists in the isolation of seminiferous tubular During vitro, sperm gradually become able to segments acrosome-react in to the from followed fractionation of response agonist, progesterone (P). Sperm spermatogenic stages VIII-XIV, by detergent-solubilized specimens must lose unesterified cholesterol to become responsive, but other a 33/50%' in the by ultracentrifugation using Percoll gradient presence of a buffer that stabilizes the aspects of the process are not well understood. These experiments integrity of microtubules and microtubular-associated proteins. Samples resulting from the frac- tested the possible involvement of intracellular pH (pHi) in the tionation procedure were monitored by whole-mount electron microscopy after negative staining, acquisition of responsiveness. Sperm were incubated 24 hr in vitro in a indirect immunofluorescence using tubulin antisera, and two-dimensional PAGE and immunoblot- modified Tyrode's medium containing 26 mg/ml bovine serum albumin in ting using antisera to a and P.,tubulin isotypes, tyrosinated and acetylated a tubulin, actin, the absence (Con) or presence (Cho) of 1 pM cholesterol (to inhibit cytoplasmic dynein, Sak57 (for spermatogenic cell/sperm-associated keratin, Mr 57 kDa/pl 5.0- sperm cholesterol loss and development of acrosomal responsiveness). 5.9) and TBP-1 (for tat-binding protein-1). We have previously reported that Sak57 is a soluble Trimethylamine (TMA, 20 mM) + P (1 g/ml) induced 21 ± 4% (mean acidic keratin associated with microtubules of the manchette (Kierszenbaum et al.; Tres & ± sem, n = 3) of Cho sperm to acrosome-react, compared to 2 ± 1 % Kierszenbaum Molec. Reprod. Dev., in press). TBP-1, a protein regarded as a transcription activa- with P alone. NH4CI and monensin gave similar results. Con sperm to P increased ± ± tor, also contains an ATPase-like domain (Rivkin et al., submitted for responding from 26 2% to 42 6% in the presence publication). Immunoblotting of TMA. In the size of the incremental experiments demonstrate the presence in fractionated manchettes of a dose-response studies, population tyrosinated tubulin, 0r._ of Cho sperm responding to TMA + P compared to P alone always and several microtubule-associated proteins, including Sak57, TBP-1, cytoplasmic dynein and i matched the incremental population of Con sperm, suggesting that TMA act/n. iactin was visualized by immunogold electron microscopy in association with microtubules was not overcoming inhibition by high cholesterol, but rather permitting of the manchette (steps 14-17), flanking the paraaxonemal-aligned mitochondria along the middle response of a special subset of sperm. Freshly ejaculated sperm did not piece of the tail.Whole-mount electron microscopy demonstrated microtubules associated with respond to TMA + P. The results suggest that elevated pH. can increase structures regarded as microtubule-associated proteins. Results of this study indicatethat this newly acrosomal responsiveness of incubated sperm, but it is probably not developed procedure for the isolation of manchettes should facilitate an understanding of the sufficient to make fresh or cholesterol-inhibited sperm responsive. molecular interaction between tubulin isotypes, cytokeratins and functional proteins during sperm Further, high sperm cholesterol does not prevent all time-dependent nuclear shaping events. changes related to acrosomal responsiveness. Supported by NIH grant Supported by USPHS grant HD1 1884 HD30763. Sunday. Sperm and Spermatogenesis (713-718) 123a 713 714 INCREASED ACTIViTY OF SPERM-ASSOCIATED HEXOS- EXPRESSION OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S AMINIDASE, AN ENZYME THAT IS REQUIRED FOR EFFI- (GAPD-S) IN CONDENSING SPERMATIDS. ((D.A. O'Brien ', J.E. Welch 2, D.O. CIENT PENETRATION OF THE EGG ZONA PELLUCIDA. Bunch 3, P.L. Magyar ', P.R. Brown3 and E.M. Eddy 3)) 'Laboratories for Reproductive [J. C. Hall, S. L. Foster, C. E. Tubbs, Y. Li, and S. Ashraf), Biology, University ofNorth Carolina, Chapel Hill, NC 27599. 'US EPA, Research Department of Biochemistry, North Carolina State University, Triangle Park, NC 27711. 3NIEHS, NIH, Research Triangle Park, NC 27709. Raleigh, NC, USA 276951. Gapd-s encodes a unique form of glyceraldehyde 3-phosphate dehydrogenase that is Previously, we reported unusually high activity levels of the expressed only during the haploid phase of spermatogenesis and may serve a pivotal glycosidic enzyme, N-acetyl--D-hexosaminidase (EC 3.2.1. 52) role in regulating glycolysis in spennatids and sperm (Welch et al., 1992; Biol Reprod associated with rat epididymal tissue, luminal fluid, and sperm cells 46:869). To examine the appearance and localization ofthe GAPD-S protein in these [Hall, J. C. et al., Biol. Reprod. 54: 914-929 (1996)]. Data from the cells, specific antisera were prepared against synthetic peptides corresponding to amino previous study provided evidence to support the hypothesis that the acids 158-194 and to amino acids 293-306 of the deduced mouse GAPD-S sequence. enzyme is involved in the maturation of sperm within the mammalian The peptides were conjugated to bovine thyroglobulin and injected into rabbits for epididymis as well as the fertilization process. In the present study, antisera production. Reactivity and specificity of the antisera were analyzed by fluorogenic substrates were used to measured and differentiate the immunohistochemistry on paraffin sections of Bouin's fixed mouse testis and activity of the A and B isozyme. While differences in the activity epididymis. Antisera raised against both GAPD-S peptides showed no detectable ratio of the A and B isozymes in sperm obtained from different staining in round and elongating spermatids (steps 1-1 1). GAPD-S immunoreactivity regions of the epididymis were observed, only the B isozyme was was first detected in steps 12 and 13 and continued to increase in intensity during steps found in the acrosome of mature sperm. Selective inhibition of the B 14 and 15. In step 16 spermatids, immunoreactivity was present in the flagella and isozyme ia abolished binding and penetration of the egg zona yit cytoplasm directly adjacent to the tubule lumen, where these cells are aligned in pellucida by sperm. The results of this study demonstrate that the B preparation for release from the seminiferous epithelium. Flagella of spermatozoa in isozyme is required for efficient binding and penetration of the egg the epididymis also were intensely labeled with GAPD-S antisera. zona pellucida by rat sperm and suggest that the B isozyme Immunoreactivity is was completely blocked by pre-incubation of each antiserum with the selectively bound by sperm and incorporated into the acrosome as appropriate peptide antigen. Antisera to both peptides also showed the same stage specificity on they mature. The significance of these findings relative to the sections from rat testes, with immunoreactivity present in maturation of sperm, fertilization process, and development of a only condensing spermatids. "potential" male contraceptive is work is These results suggest that GAPD-S may be subject to translational regulation, since the discussed. [This supported is not detectable until well after levels of by the National Science Foundation grant # IBN: 9511822 protein steps 12-13, peak Gapd-s mRNA are awarded to Dr. Joseph C. Hall.] reached at step 9 of mouse spermiogenesis (Mori et al., 1992; Biol Reprod 46:859).

715 716 GERMLINE STEM CELL IDENTITY AND THE MOLECULAR- A Testis-specific Protein Binds to the Putative Y-box Within the GENETIC BASIS OF THE DROSOPHILA ZK MUTANT. ((A.A. Kiger Promoter of the Mouse Testicular Cytochrome c Gene and M.T. Fuller)) Department of Developmental Biology, Stanford School of Medicine, Stanford, CA 94305. Gary K. Yiu', Mary T. Murray2, and Norman B. Hecht' 'Department of Biology, Tufts University, Medford, MA 02155, USA A unique feature of stem cells is their ability to divide asymmetrically 2Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48202, and self-renew. During spermatogenesis, germline stem cells USA continuously support the repopulation of the testis with differentiating Two distinct isoforms of cytochrome c are found in mouse testis, gametes throughout adult male life. Drosophila melanogaster males namely the somatic form (cyt. cs) and testicular form (cyt. cT). The cyt. cs homozygous for a male sterile allele of wonder kloten (zk) have tiny present in germ cells in early stages of spermatogenesis is gradually replaced testes due to the absence of germline, suggesting that the gene may by cyt. CT during meiosis. To study how the testis-specific transcription of be required for establishment or mainenance of stem cell function in cyt. cT is mediated, we have examined DNA-protein interactions in its males. Antibody staining of zk mutant embryos reveals that germline proximal promoter. By DNase I footprinting, we found that nuclear proteins pole cells are present in the embryonic gonad, and a few from both mouse liver and testis nuclear extracts bound to a sequence at -18 morphologically distinct spermatocytes are occasionally observed in to +31. This region contains the binding sites of SPI and CTF/NF-I, as well the abnormally small zk larval testis. However, markers for germline as a putative Y-box protein binding site that is present in many germ cell- stem cells are absent in the adult, implying that the early zk germline specific promoters. Competition mobility shift assays revealed that the Y-box fails to fully differentiate or be retained as stem cells in the adult sequence, but not the SPI or CTF/NF-I binding sites, is essential for protein gonad. The zk phenotype is associated with a P-element insertion, and the flanking genomic DNA has been cloned. Developmental binding to the cyt. CT -65/+40 fragment. Southwestern blots and Northem analysis has identified several candidate transcripts in the immunoprecipitations have established that the mouse testicular 52 kD zk region, and in situ hybridization of genomic probes to late embryos homologue of the Xenopus germ cell-specific Y-box protein binds at -13 to recognizes transcripts in the gonad and central nervous system. -2 and a 50 kD protein present in both liver and testis nuclear extracts also zk allelism with independent P-element insertions and EMS-induced binds to this region. These data demonstrate that different nuclear proteins in mutations demonstrates that zk is an essential gene also required for liver and testis are competing for binding sites containing the putative Y-box. male germline development. We propose that this protein-DNA interaction is important for the transcription of the mouse cyt. cT gene during spermatogenesis.

717 718 COMPARATIVE SPERM HISTOCHEMISTRY IN SEVERAL SPECIES CULTURE OF RAT SEMINIFEROUS TUBULES AS A TOOL FOR TOXICOLOGICAL STUDIES.((C.B.Loaballo and K.A.H.Dolder)). OF ANURA ((Mauricio J.L.V. do Amaral 1,3, Fernando Ananias', AdAo J. Department of Cellular Biology. Biology Institute. Cardoso2 and Shirlei M.Recco-Pimentel)) 1Dept. Cell Biology and 2Dept of Universidade Estadual de Caapinas, CP.6109 - 13083-970. Zoology State University of Campinas, CP6109, 13083-970 Campinas SP, and Caapinas/SP, Brazil. (Spon. by A.Sesso) 3Dept. of Cell Biology, Embriology and Genetics - Federal University of Santa The conditions of seminiferous tubule cultures were in Catarina, 88040-970 Florian6polis SC, Brazil. (Spon . by A . T. Ya-ada) established order to use the system for testing the action of cadmium on the seminiferous epithelium. Different fragmentation conditions, manipulation, culture medium and Sperm of three species of Physalaemus, five of Hyla, one of supplements were tested. The tecidual disturbances caused by the culture Leptodactylus, and one of Brachycephalus, were compared by cytochemical techniques were established to be used as a control situation. The main alteration methods, using toluidine blue (TB) and alkaline fast green (AFG). The sperm of observed was related to the organization of the epithelium. The techniques the majority of the species, showed TB metachromasy, except the L. podicipinus involved caused dislocation of germ cells directed towards the tubular lumen. The sperm, which exhibited a green color.These results suggest a higher chromatin epithelial integrity is fundamental for cellular development. As a consequence compactation in the L. podicipinus sperm. Excepting P. gracifis, the sperm of all degeneration signs could be observed during the culture period in a progressive species stained with AFG, even after deamination. These results suggest the pattern. The seminiferous tubule analysis involved light microscopy and scanning presence of arginine in the basic sperm protein composition. The differences electron microscopy. A three day period was set for the experiment with daily among species pointed out indicate that the rate of arginine/lysine is higher in L. fixations. Three concentrations of cadmium (1, 10 e 100p&M) were tested during a podicipinus than in the other species. The P. graciis sperm, however, do not stain one hour period of exposition. Another experiment with direct exposition of with AFG after deamination, suggesting that their basic sperm proteins consist tubules to the metal (1pM) was cried out during the entire culture period. As a basically of lysine. Thus, following Bloch's (1969) classification, the basic sperm consequence of cadmium present in the culture, degeneration was intensified and protein of P. gracifis would be classified as a Rana type and the other species as a accelerated. Cadmium caused progressive degenerative alterations, proportional to Mytilus type These findings are not consistent with the phylogenetic relationships the doses applied andto the time of exposition. This study shows that cadmium currently accepted for the species studied. has a cytotoxic effect directly on the germ cells and therefore the testicular Supported by CAPES/PICD degeneration observed "in vitro" after contact with this metal, is not due onlyto injury of testicular vasculature, as has been considered previously from studies carried out "in vivo". CAPES Masters Fellowship 124a Sperm and Spermatogenesis (719-724). Sunday

719 720 RAB3A PROTEINS ARE PRESENT IN THE ANTERIOR HLA CLASS EXPRESSION IN HUMAN TESTIS. ((H. Hutter, H-L. Chen*, HEAD OF MOUSE SPERMATOZOA. ((C.R. Ward and J.S. Tash", B. Uchanska-Zieglere, A. Ziegler+, A. Blaschitz, G. Dohr and S.N. Martin)) Department of Clinical Studies, University of J.S. Hunt*)) Dept. of Histology and Embryology, KF-University of Graz, 8010 Pennsylvania School of Veterinary Medicine, Philadelphia, Graz, Austria; *Dept. of Anatomy and Cell Biology, 'Dept. of Physiology, PA 19104-6010. University of Kansas Medical Center, Kansas City, Kansas 66103, USA; Sperm acrosornal exocytosis as induced by the egg- +Inst. for Experimental Oncology and Transplantation Medicine, Free associated extracellular matrix glycoprotein, ZP3, is an University of Berlin, 14050 Berlin, Germany example of regulated exocytosis. Rab3 proteins play an important role in regulated exocytosis in somatic cells. In The expression and function of MHC class antigens in human order to define a potential role for rab3 in ZP3-induced spermatogenic cells has been a matter of long dispute. Since there is still a acrosornal exocytosis, we attempted to determine the controversy over the presence of HLA class mRNA and protein on human presence and identify the cellular localization of rab3 in male gametogenic cells we investigated the expression HLA class mature, mouse sperm. Western blots of detergent extracts of molecules by immunohistochemistry using a panel of monoclonal antibodies cauda epididymal sperm were incubated with rab3a (mAb) to HLA class antigens. Using mAb recognizing HLA class I heavy antibodies. The bound to a kDa antibody specifically 23,500 chain/f32-microglobulin complexes no expression of these complexes could protein, and binding was inhibited by antibody pretreatment be detected. However, several mAb recognizing classical and non-classical with the peptide against which it was generated. Using indirect immunofluorescence, rab3a localized to the anterior HLA class heavy chains only (HCA2: anti-HLA-A, -G; HC10: anti-HLA-B, LA45: showed a reaction on and head of the sperm. Therefore, we conclude that rab3a is -C; anti-HLA-A, -B), positive primary present in the head of mouse sperm and that it may play a secondary spermatocytes. In addition, the expression of the non-classical regulatory role in ZP3-induced acrosomal exocytosis. class HLA-G gene on spermatogenic cells was analyzed by in situ hybridization using a specific Digoxigenin-labeled oligonucleotide. These experiments revealed the presence of HLA-G mRNA in primary and secondary spermatocytes, whereas in other stages of sperm cell maturation e.g. spermatogonia, spermatides and mature sperm cells no mRNA could be detected. These results were supported by quantification on a cell imaging system. Our data suggest that in human tests HLA class heavy chain expression is dependent on the location and the differentiation state of the spermatogenic cells during maturation.

721 722 REGULATION OF SPERM MOTILITY AND TYROSINE PHOSPHOR- OSHO, A GENE REQUIRED FOR GERM LINE STEM CELL MAINTENANCE IN YLATION BY PKA ANCHORING INHIBITOR PEPTIDES. ((S. DROSOPHILA. ((M. Fogarty, C. Wood and M.T. Fuller)) Stanford University, Department of Developmental Biology. Stanford, CA 94305 Vijayaraghavan 1, S.A. Goueli2, M.P. Davey3, and D.W. Carr3)). Oregon Primate Research Center, Beaverton, OR1; Promega Corporation, Madison, I am studying a Drosophila male sterile mutation called one shot (osho) that appears to WI2; Veterans Affairs Medical Center, Portland, OR3. affect the maintenance of germ line stem cells. In wild type Drosophila spermatogenesis stem cells at the apical tip of the testis divide asymmetrically giving rise to a new stem Cyclic AMP-dependent protein kinase (PKA) is anchored at specific cell, and a genial cell that goes on form 64 mature sperm. A timeline of sperm subcellular sites via the interaction of the subunit with A-kinase development is visible in the testis, with the younger cells near the apical tip and the more regulatory (R) the of the testis. a via an helix motif. mature cells further down length Wild type males eclose with large anchoring proteins (AKAPs) amphipathic binding number of mature sperm bundles and all of the younger stages in spermatogenesis Synthetic peptides containing this amphipathic helix domain competitively present. In contrast, newly eclosed osho males have only a handful of mature sperm disrupt PKA binding to AKAPs and cause loss of PKA modulation of cellular bundles and a striking lack of any younger cell types. This implies that the stem cells are responses. We have recently shown that S-Ht3 1, a cell permeant anchoring initially able to divide and that their daughter cells are able to complete the inhibitor peptide (AIP), is able to arrest mammalian sperm motility. In this spermatogenesis program, however at an early and specific point in development, the report we identify two tyrosine phosphorylated proteins whose stem cells stop dividing and no new sperm is made. The original osho allele was caused phosphorylation levels are regulated by S-Ht3 1. The phosphorylation levels by the insertion of a transposable element that I have used as a molecular handle to clone of one of these proteins, a 55 kDa protein found in the soluble fractions, the genomic DNA in the presumptive osho region. I have isolated a number of cDNAs in correlates with the level of sperm motility. Immotile caput sperm have low the region and am in the process of defining candidate genes through northern analysis levels of tyrosine phosphorylation of this protein compared to vigorously and in situ hybridization. motile caudal sperm. Agents which increase motility such as IBMX and 8-Br- cAMP increase tyrosine phosphorylation of the 55 kDa protein while agents which decrease motility such as S-Ht31 and ionomycin plus calcium completely abolish tyrosine phosphorylation of the 55 kDa protein. This is the first documentation of a protein whose tyrosine phos-phorylation parallels motility. Treatment of sperm with S-Ht3 1 also affects the tyrosine phosphorylation of a 39 kDa protein found in the insoluble fraction. In this case, S-Ht3l causes a dramatic increase in phosphorylation, suggesting that sperm PKA anchoring affects a substrate specific tyrosine kinase and/or phosphatase which may be involved in the regulation of sperm motility. Supported by NIH HD-30908 (SV) and HD-32508 (DWC).

723 724 MOLECULAR CLONING AND STRUCTURAL CHARACITERIZA- TISSUE SPECIFIC ALTERNATIVE SPLICING OF MURINE TION OF MURINE SULFATED GLYCOPROTEIN-1 SGP-1 (PROSAPOSIN) GENE. ((N. Hay, Q. Zhao, and C.R. (PROSAPOSIN) GENE. ((Q. Zhao, and C.R. Morales)) Department Morales)) Department of Anatomy and Cell Biology, McGill of Anatomy and Cell Biology, McGill University, Montreal, Quebec, University, Montreal, Quebec, Canada, H3A 2B2. Canada, H3A 2B2. Sulfated glycoprotein-l exists in two forms. A 65 kDa protein Sulfated glycoprotein-l (SGP-1), also known as prosaposin, is a known as prosaposin, which is the lysosomal precursor of smaller multifunctional protein involved in glycosphingolipid degradation and lysosomal saposins involved in glycosphingolipid degradation and a lipid transport. SGP-I (prosaposin) may also play important roles in the 70 kDa protein which is secreted into the lumen of the seminiferous reproductive system as well as in the nerve system. SGP-1 exists as a tubules. In the human, four saposins termed A, B, C and D exist in 70 kDa secreted glycoprotein or as a 65 kDa lysosomal precursor of the lysosomes. The alternative splicing of the prosaposin gene four smaller saposins. In the human it was shown to exist as an integral results in the inclusion or exclusion of exon 8. Consequently, exon component of the plasma membrane of neuronal cells. In this study we 8 encodes for three amino acid residues (Gin-Asp-Gln) which may used a PCR-based method in conjunction with a 32P-labeled SGP- 1 be present or absent in the saposin B domain. We have recently cDNA probe to double screen a mouse genomic library. Several positive cloned the murine SGP-l (prosaposin) gene and found that it also clones were selected and purified to homogeneity. DNA sequencing of contains exon 8. In the present study we have used reverse the largest clone revealed that the whole SGP-1 genomic DNA is about transcription-polymerase chain reaction (RT-PCR) to study the 25 kb in length. It contains a total of 15 exons ranging from 9 bp to distribution of each alternatively spliced mRNA in several tissues. 1,000 bp, and a total of 14 introns ranging between 89 bp and 15,000 RT-PCR showed that a predicted fragment of 115 bp containing bp in length. The intron-exon organization corresponds to the AG/GT exon 8 was found in brain, heart and muscle, and 106 bp fragment concensus sequences. The regions encoding saposins A and D include 3 without exon 8 was found in kidney, spleen, pancreas, lung and exons, while the regions encoding saposins B and C include 4 and 2 testis. A weak 106 bp fragment was also found in brain, heart and exons respectively. The sequencing of the 5' upstream region of the muscle. Our results didn't exclude the presence of an intermediate SGP-I gene as well as the 15 Kb large intron preceding exon 2 are still form containing 6 bp of exon 8 in brain, heart and muscle. The GIn- in progress. The cloning of the gene will permit the targeting of Asp-Gln insertion was indicated to abolish the capacity of saposin mutations by homologus recombination and site directed mutagenesis B to bind GM, ganglioside and to increase its affinity for sulfatide and to generate mouse models deficient in saposins. Supported by and sphingomyelin. Therefore, these results suggest that alternative MRC. splicing may confer shingolipid specificity to saposin B according to the sphingolipid abundance in different tissues. Supported by MRC. Sunday. Sperm and Spermatogenesis (725-730) 125a 725 726 GM2 ACTIVATOR PROTEIN EXPRESSION AND LOCALIZATION IN THE MALE HORMONAL INDUCTION OF SPERM RELEASE IN ANURANS AND REPRODUCTIVE TRACT OF ADULT NORMAL AND HEXOSAMINIDASE A- STRUCTURE OF Lepidobatrachus laevis SPERM. ((W. L. Wapgener and E. J. DEFICIENT MICE. ((H.I. Adamali, D. Mahuran, J-Q. Huang, R.A. Carroll, Jr.)) eartment of Biology, University of Califomia at Riverside, Gravel, J.M. Trasler, L. Hermo)) Departments of Anatomy & Riverside,UA 21521. Cell Biology, Pediatrics and Human Genetics and Pharmacology, Sick McGill University, Montreal, Quebec, and Hospital for Most developmental studies using amphibia have required testicular macerates Children, Toronto, Ontario. in order to effect timed in vitro fertilization. A significant limitation of this approach is that the genetic contribution of the male cannot be studied in The Gm2 activator protein is a required cofactor for the subsequent Human gonadotropin hydrolysis of GM2 ganglioside by the lysosomal enzyme experiments. releasing hormone (GnRH) hexosaminidase A (Hex A). In this study, mRNA levels of the injections were used in Lepidbaftchus laevis, Xenopu-s laevis and Rana to stimulate sperm activator were quantitated by Northern blot analysis, and pfpens maturation. Sperm were expressed from each of protein was localized immunocytochemically using anti-G these species within a few hours post injection either by mechanical stimulation activator antibody, in the testis and epididymis of normal or cloacal ravage. Typical volumes of 'ejaculate' varied from 8 to 3700 tuL and adult mice (control). Tissue of hexosaminidase A-deficient sperm cell densities ranged from 0.4 to 395xl0sperm/mL. The sperm mice (Hexa-l-), produced by targeted disruption of Hexa gene, obtained using this method were viable, ferilized eggs In vitro and produced were examined to test whether absence of hexosaminidase A normal tadpoles in the case of Lepdobatrachus haevl& The structure of could alter GM2 activator protein expression. mRNA levels of Lepdbtaachus laevis sperm was studied by phase contrast and electron the GM2 activator protein in both testis and epididymis of microscopy. Observations at the light level of mature sperm showed an Hexa-/- mice were above control steady state levels. In the acrosomal segment of 5.4±0.9pm, followed by a midpIce of 12.3±0.8 im. The testes of control mice a fine granular immunoperoxidase tail was 54.9 ± 3.4 pm long and appeared to have two lateral axial fibers and a reaction was observed in mid-pachytene spermatocytes, Sertoli central undulating membrane. At the electron microscopic level, a thin and cells. In contrast in the Hexa-/- mice cells interstitial acrosome was followed posteriorly by a typical sperm nucleus. The centriole all germ cells and especially spermatocytes and spermatids, structure consisted of one proximal centriole and one distal centriole. Two and a dramatic increase in Sertoli interstitial cells showed axonemes were elaborated from the distal The tall also reactivity. In the control epididymis, principal cells of centriole. contained two axial fibers about in diameter medial to the axoneme and connected by the initial segment and cauda regions were weakly reactive 0.3pm the undulating membrane. The calcium A23187 an whereas the reaction was more pronounced in the caput and ionophore induced acrosome reaction based on electron microscopic observations. corpus regions. In the Hexa-/- mice the pattern of staining This hormonal method of anuran sperm collection will provide a convenient non-injurious was similar to control animals however the intensity of the way to obtain anuran for basic studies of immunoperoxidase was highly increased in principal cells. sperm reproduction and development. Clear cells were weakly reactive in both animals. The distinct cellular and epididymal region-specific expression in control mice as well as the elevated levels of mRNA and protein in the Hexa-/- mice suggests regulated expression of the GM2 activator in the testis and epididymis.

727 728 CLONING AND CHARACTERIZATION OF THE 0-MER RECEPTOR TYROSINE GERM CELL-SERTOLI CELL ADHESION INVOLVES KINASE IN THE MOUSE TESTIS ((C.A. Dowds+, E.M. Eddy*. P.M. Saling+)) FUCOSYLTRANSFERASES (1Ts) AND THEIR CARBOHYDRATE +Departments of Cell Biology and Obstetrics and Gynecology, Duke LIGANDS. ((S.S. Raychoudhury, and C.F. Millette)) Department of Cell University, Durham North Carolina 27710 Biology & Neuroscience, University of South Carolina School of Medicine, ^NIEHS, NIH, RTP, NC 27709 Columbia, SC 29208.

Murine c-mer is a receptor tyrosine kinase whose message is found in We have identified multiple fucosyltransferases [a(1-2)-, a(1-3)-, a(I-4)- many tissues, including the testis. In examining the role of this receptor FTs] on cells of the rat seminiferous epithelium as demonstrated by fucose in the testis, we have cloned the mouse c-mer from testis and germ cell incorporation into phenyl-j3-D-galactoside, 2'-fucosyllactose and lacto-N- specific cDNA libraries, and a composite of two overlapping clones fucopentaose-I, respectively (Raychoudhury & Millette, Biol. Reprod. contains 5' untranslated sequence, the complete coding region, and a 51:1006-1013,1994). Now, using fluorescence laser scanning cytometry, complete 3' untranslated region, including a poly A+ tail. Northern we have established that multiple FTs and their ligands are involved in germ analysis identified a predominant band at 4.5 kb, as had been seen cell-Sertoli cell adhesion. Sertoli cells were isolated from 20-day old CD rat previously (Graham, et al., Oncogene 10, 2349-59). Testicular testis and cultured for 6-10 days. Mixed germ cells were isolated by maturation in prepuberal mice involves proliferation and differentiation of enzymatic dispersion of adult rat testis and cultured overnight before both germ cells and somatic cell types. More detailed analysis of mouse labeling with 10 pM calcein-AM as fluorescent indicator. ACAS (adherent testis RNA indicates that mouse c-mer is expressed early, on or before cell analysis and sorting) 570 laser cytometry was used to determine the day 8, and remains on through day 30 and into adulthood. Interestingly, number of bound germ cells on the Sertoli cell monolayers in the presence while Sertoli cells stop their proliferation at day 16, and cells undergoing or absence of a variety of low molecular weight acceptors for fucose. Co- meiosis are found only starting at day 10, spermatogonial cells continue to incubation of labeled germ cells with Sertoli cell monolayers in the presence proliferate beginning from around day 6 throughout the reproductive life of GDP-fucose, Lewis-X and 3'-sialyl-Lewis-X oligosaccharides resulted of the mouse. Germ cells are also in intimate contact with Sertoli cells in significant inhibition of germ cell binding (approximately 50%) when throughout this time. To further detail mer's localization and role in the compared to the untreated controls and to control samples incubated with testis, analysis of RNA from various isolated testicular cell types is cellobiose, melibiose anda-D-mannopyranoside which do not serve as underway, as is the production of antipeptide antibodies specific to mer's fucose acceptors. Our results suggest that multiple Frs and the extracellular domain. Supported by NIH HD18201 and HD29125. lectin/selectin ligands are implicated in germ cell Sertoli cell adhesion and thus help regulate germ cell adluminal translocation within the seminiferous tubule. Supported by HD-27581 (CFM).

729 730 EXPOSURE TO THE ENVIRONMENTAL POLLUTANT, EVIDENCE FORTHE INVOLVEMENT OFSYNTAXIN, OCTYLPHENOL, CAUSES SPERM DEFORMITIES AND SHRINKAGE SYNAITOTAGMIN, AND SNAP-25 IN REGULATING ACROSOMAL OF TESTES AND MALE ACCESSORY SEX ORGANS. ((F.R. EXOCYTOSIS. ((J.R. Schulz, J.D. Sasaki, and V.D. Vacquier)) MBRD, THE SIO, UCSD, CA. 92093-0202 Boockfor, and C.A. Blake)) Department of Cell Biology and Columbia, Neuroscience, University of South Carolina School of Medicine, During sea urchin fertilization, contact with the outer investments of the egg SC 29208. signals the calcium-triggered exocytosis of the sperm acrosomal vesicle resulting in release of the gamete recognition protein bindin. To investigate The widespread environmental toxicant, 4-tert-octylphenol (OP), has been how this event is regulated we used a degenerate primer PCR based approach shown to have potent biological effects on mammalian cells in culture, to identify sequences encoding the sea urchin homologues of the exocytosis regulating proteins syntaxin, synaptotagmin and SNAP-25 from an many of which appear to be estrogenic in nature. Very little information S.purpuratus testis cDNA library. To confirm the presence of syntaxin in is available on the influence of this in vivo, on cells compound especially spermatozoa an antiserum generated against the S.purpuratus egg form of and tissues well recognized as sensitive to environmental influences, such syntaxin was used (kindly provided by Dr. G.M. Wessel). This antiserum as those in the male reproductive system. In this study, we injected OP recognized a single form of syntaxin from sperm plasma membranes on (20 or 80 mg) or estradiol (E; 0.8 or 8 #g) in oil s.c. into adult male rats western blots that co-migrates with syntaxin present in sea urchin egg cortical 3 times a week for either 1 or 2 months. We found that an 80 mg dose of granule lawns. Cell fractionation of sea urchin spermatozoa indicate that syntaxin localizes sperm head. OP and one or both doses of E greatly reduced sperm numbers, as well as exclusivelyto the Immunofluorescence confirms an intracellular location over the acrosomal vesicle. the sizes and weights of the testes, epididymides, prostate glands, seminal exclusively These results suggest a role for these proteins in regulating acrosomal vesicles, and coagulating glands. A marked increase in the proportion of exocytosis in sperm similar to synaptic vesicle exocytosis at the pre-synaptic head and tail sperm morphological abnormalities were also observed with membrane. (supported by NIH grant 12986 to VDV). these treatments and with the lower dose of OP administered for the 2- month period. Our results clearly demonstrate that OP can severely reduce the size and/or function of most, if not all, of the male gametogenic and accessory reproductive organs. Moreover, the similarity in these cell and tissue changes between rats treated with OP and those treated with E suggest strongly that OP may exert its action in an estrogenic-like manner. Supported by NIH grant HD 22687. 126a Sperm and Spermatogenesis (731-735). Sunday 731 732 SPERMATOGENESIS IN EXORISTA SORBILLANS (Diptera: Percoll in a non-toxic flowcytometric assay to separate viable from Tachinidae) as revealed by Light and Electron Microscopy. ((H.R non-viable sperm. ((J.Stap, R.A.Hoebe, S.Merton, P.J.M.Bakker, Puttaraju and H.B. Manjunatha)) Department of Sericulture, Jnana J.A.Aten.)) Laboratory for Radiobiology Bharathi, Bangalore University, Bangalore - 560 056, India. In the described assay the DNA of cryopreserved bull sperm is stained with the vital stain Hoechst 33342. The stained sperm cels are analysed using a The events of spermatogenesis in a single follicle testis of flowcytometer/cellsorter. We found that Percoll can be used to quench the Exorista sorbillans are in a sequential fashion starting from distal to Hoechst fluorescence of the dead cells. By adding Percoll to the stained cell proximal end. A total of 256 spermatozoa are formed from six suspension the position of the fluorescence distribution of the dead cells is synchronous mitotic divisions followed by melotic division of moved away from the position of the distribution of the living cells. In this spermatogonium. Phenominal~morphological changes of spermatid way it is possible to determine the fraction of dead cells. Furthermore, by Into spermatozoon are described. The reduction division and sorting out cells from the fluorescence distribution with the highest intensity, spermatid into spermatozoon are described. The reduction division the fraction of viable cells can be enriched fron 40% in the unsorted sample and spermatid formations are more in 6 d frequent pupal stage. The to 85% in the sorted population in the case of cryopreserved sperm. The entire process of spermatogenesis completed in 10 d pupal stage. viability of the selected and sorted sperm cells was determined independently The ultra structure of sperm exhibits 9 + 9 + 2 configuration of by Propidium Iodide exclusion. axoneme and in equal sized nebenkern derivatives.The curling off of outer membrane starts from the region of opening of vacuole and the appearance of two whorls of sloughed off nuclear membrane at the lateral surface of the sperm appears to be species specific.

733 734 A DEVELOPMENTAL STUDY OF RAT SPERM a-L-FUCOSIDASE GENETIC AND PHENOTYPIC CHARACTERIZATION OF THE SPE-25 GENE DURING EPIDIDYMAL MATURATION ((I. Abascal', M. Aviles', J.A. OF C. ELEGANS ((Department of Biological Sciences, Humboldt State University, CA 95521 Martinez - M.T. Skalaban J. Arcata, Enter abstract text (do NOT enter title, author, inst again): Menarguez', Castells', S.R. , Ballesta' and Mutations J.A. Alhadeffb.)) 'Department of Cell Biology, Medical of in the spe-25 gene result in sterility of both hermaphrodites and males. School, University The sterile phenotype is due to the inability of the mutants to produce spermatids. Murcia, Spain and bDepartment of Chemistry, Lehigh University, In spe-25 mutants, defective spermatocytes fail to undergo meiosis. Even though Bethlehem, PA, USA 18015. the defective cells enter meiosis, they fail to complete prophase I. Temperature shift Previous studies of our group reported the occurrence of a novel o-L- experiments using a temperature sensitive allele (hcl32ts) have clearly shown the fucosidase associated with the rat sperm plasma membrane. Light and electron temperature sensitive period to be the late L3 early L4 stage of development. This microscopy localized the fucosidase on the convex region of the sperm head. is consistent with the observed phenotype of the defective spermatocytes. By two This enzyme is different from other mammalian fucosidases, i.e it is membrane- factor and deficiency mapping, the spr-25 gene has been located to the left arm of chromosome III, 0.3 mu from unc-36 between the nDf2O and nDfl6 deficiency associated, it has a neutral pH optimum, and it is not In this lysosomal. study, breakpoints. DNA transformation rescue analysis of the sterile biochemical techniques have been used to characterize this in differ- phenotype is in enzyme progress. Using an F1 non-complementation screen, two new alleles of the spe-25 ent parts of epididymis: caput, corpus and cauda. The pH activity curves for gene have been identified. Characterization of these alleles is in progress. ci-L-fucosidase solubilized with 0.5M NaCl from each part of the epididymis (Undergraduate research project supported by HHMI 71194-535001, NIH resulted in two peaks with an optimum near pH 7.0 and a second minor opti- R15GN52679-01 and.)) Department of Biological Sciences, Humboldt State mum, with about 40% of maximal activity, between pH 3.3-3.7. These results University, Arcata, CA 95521 are comparable to the pH optimum curves from intact sperm. SDS-PAGE and Mutations in the spe-25 gene result in sterility of both hermaphrodites and males. Western blotting indicated that the NaCl-solubilized enzyme from the three The sterile phenotype is due to the inability of the mutants to produce spermatids. In spe-25 mutants, defective spermatocytes fail to undergo meiosis. Even different epididymal zones contained two protein bands near 5OkD and 54kD, though the defective cells enter meiosis, they fail to complete prophase I. Temperature shift are to antifucosidase which highly immunoreactive antibodies. The isoelectric experiments using a temperature sensitive allele (hcl32ts) have clearly shown the focusing profiles indicated some differences: while cauda sperm profiles show temperature sensitive period to be the late L3 early L4 stage of development. This is two major isoforms with pls near 5.4 and 7.0, caput and corpus only show one consistent with the observed phenotype of the defective spermatocytes. By two factor isoform with a pl near 7.0. The present results suggest that a second, more and deficiency mapping, the spe-25 gene has been located to the left arm of chromo- acidic isoform of cs-L-fucosidase is produced or expressed during epididymal some III, 0.3 mu from unc-36 between the nDf2O and nDfl6 deficiency breakpoints. maturation of rat sperm that could be related to sperm-egg recognition pro- DNA transformation rescue analysis of the sterile phenotype is in progress. Using an Fl non-complementation screen, two new cesses. Supported by a grant from DGICYT (PB-93-1123-C02-01) Spain alleles of the spe-25 gene have been identi- fied. Characterization of these alleles is in progress. (Undergraduate research project supported by HHMI 71194-535001, NIH R15GN52679-01 and Institutional grants.)

735 PHENOGENETIC ANALYSIS OF STERILITY IN HYBRIDS DROSOPHILA VIRILIS X DROSOPHILA LUMMEI. ((Lyudmila I. Falileeva.)) N.K.Koltzov Institute of Developmental Biology, RAS, 26 Vavilova St., Moscow, 117334, Russia Species of D. virilis phylad is an ideal object for studying speciation. Hybrid sterility is postzygotic isolation mechanism that is usually detected in the F1 generation, while novel combinations of genetic elements in subsequent hybrid generations can reveal incompatibility systems which are impossible to detect in Fl. The sterility of interspecific hybrid males between the sibling species Drosophila virilis and Drosophila lummei was tested in reciprocal F1, F2, F3, F4 and FB generations with different combinations of cytoplasm, autosomes and sex chromosome at different temperature conditions. Males with motile sperm were scored as fertile, males with no motile sperm were scored as sterile. When D. virilis was the mother, about 9 per cent of the F1 male progeny was sterile (temperature +250C, heterospecific chromosome 6); and more 96 per cent of the F1 male progeny was sterile (temperature +170C, hemizygous chromosome 6, heterospecific autosomes). The X chromosome of D.lummei, especially its part in loci vermilion (v, 1-25.5) caused sterility in 90 per cent of FB1 males in the presence of heterospecific autosomes. Our results emphasize the main role of the X chromosome, Y chromosome, autosomes 2 and 6. Spermatozoons of FB1 hybrid sterile males and D.virilis fertile males have been examined by electron microscope. Electron microscope analysis has shown that nebenkern and its membrane in sterile males sperm had abnormal morphology. Sterile male spermatozoon membranes had many breaks. We have concluded that this abnormality was the main cause of sterility in hybrid males. Sunday. Neural Development I (736-741) 127a

736 737 ACIDIC CALPONIN AND REGULATION OF NEURONAL PROCESS THE EXPRESSION OF Id GENES ENCODING TRANSCRIPTIONAL OUTGROWTH. ((J.M. Willard, D.D. Murphy and S. Beushausen)) INHIBITORS IS REGULATED DURING IN VITRO ASTROCYTE Laboratory of Neurobiology, NINDS, NIH, Bethesda, MD 20892. DIFFERENTIATION. ((P.J. Andres-Barquln, M.C. Hemandez, T. Hayes*, R. McKay', and M.A. Israel)) Department of Neurological Surgery, School of Acidic calponin (AC) is a 36 kDa actin-binding protein that participates in Medicine, University of Califomia, San Francisco, CA 94143, and *Laboratory several aspects of signal transduction pathways involving tyrosine kinases, of Molecular Biology, National Institute of Neurological Disorders and Stroke, GTP binding and cell-cycle directed ubiquitin-mediated proteolysis. Levels National Institutes of Health, Bethesda, Maryland 20892. of AC in hippocampal neurons cultured from E18 mouse embryos are Idgenes encode a class of nuclear transcription factors known as helix-loop- prominent for the first 2 days in culture, then gradually diminish. helix proteins (HLH). Members of the basic helix-loop-helix (bHLH) family act as Treatment of cultures with 25 ng/ml -of 2.5S NGF leads to an increase in the transcription factors and contain a domain of basic amino acids 5' to the HLH levels of AC concomitant with alterations in neurites. These results were motif. The basic domain of such bHLH transcription factors bind to DNA. ID corroborated in the mouse neuroblastoma cell line, N2A, which were proteins lack the basic amino acid domain necessary for DNA binding. As a assayed at various times throughout the cell cycle for AC levels and during consequence of forming heterodimers with bHLH proteins, ID proteins inhibit differentiation induced by serum-deprivation. Acidic calponin remained at DNA binding and transcriptional activation. This mechanism is considered of in moderate levels throughout the first 5 days of differentiation, then decreased responsible for the inhibition cell differentiation attributed to ID proteins to low or undetectable levels. several different tissues. To study regulation of Id genes during astrocyte differentiation we a mouse with an construct out more evaluated astrocyte precursor cell line, NESHip2- N2A cells stably transfected AC put processes 28, which was immortalized with a recombinant retroviral vector expressing the under both log phase growth and differentiation, whereas, cells stably conditional mutant SV40 T-antigen, U19tsA58. This cell line differentiates transfected with a reverse construct have fewer processes than control cells along the astroglial lineage when serum is removed from the culture medium under all conditions. and the temperature of culture is switched from the permissive to the non- In conclusion, acidic calponin may be an important controlling factor in permissive temperature. We determined that idi and id3 are highly expressed process outgrowth in neuronal cells and in neuronal regeneration. in proliferating NESHip2-28, while 1d2 and 1d4 are expressed at lower levels. Upon the Induction of differentiation, the expression of all tour Id family members initially increases dramatically, at a time when GFAP expression becomes detectable, and subsequently decreases. These results suggest that ID proteins may play a role In the control of astrocyte differentiation. P.J.A.B. is a Research Fellow of the Spanish Ministerio de Educacion y Clencia.

738 739 DEVELOPMENTALLY REGULATED EXPRESSION OF POLYSIALIC INTEGRINS MEDIATE MANGANESE- INDUCED NEURONAL DIFFERENTIATION IN ACID IN THE CENTRAL NERVOUS SYSTEM DIRECTED BY TWO PC12 CELLS. ((P.J. Gallagher, J.L. Amodeo, H. Howie, J.A. Roth' POLYSIALYLTRANSFERASES, PST AND STX. ((Jun Nakayama*+, and P.J. Lein)l) Biology, Canisius College, Buffalo, NY 14208 Kiyohiko Angata*, Edgar Ong*, Tsutomu Katsuyama+, Yasumasa Arai±, and *Pharmacology and Toxicology, SUNTAB, Buffalo, NY 14214 and Minoru Fukuda* )) From the *Glycobiology Program, The Burnham Previous studies have demonstrated that exposure to manganese Institute, La Jolla, CA 92037; +Shinshu University Hospital, Matsumoto ion (Mn) causes PC12 cells to form neurites (Lin et al., 1993, 390, Japan; and IJuntendo University School of Medicine, Tokyo 113, J. Neurosci. Res. 34:546-561). We have examined the role of Japan. integrins in this phenomenon. Integrins comprise an extensive family of aig heterodimeric receptors that mediate cellular Polysialic acid is a developmentally regulated carbohydrate mainly attached to interactions with the extracellular matrix. The affinity and the neural cell adhesion molecule (N-CAM) and is thought to modulate the specificity of these receptors is modulated by divalent adhesive properties of N-CAM. Recently, we and others have cloned cDNAs cations. Our data indicate that Mn-induced neurite outgrowth encoding a polysialyltransferase (PST) that forms polysialic acid on N-CAM. is markedly inhibited (90 1001) by antiserum that recognizes demonstrated that another forms Other studies sialyltransferase, STX, theS1 and S3 integrin subunits. Function blocking antiserum polysialic acid on a yet undefined protein(s). In the present study, we first specific for the Aim integrin subunit inhibits the neurite- In order to demonstrated that STX attaches polysialic acid to N-CAM. promoting activity of Mn by 67-871, but has no significant neural determine the roles of PST and STX in polysialic acid synthesis during effect on cell adhesion to the substrate. SDS-PAGE analysis of development, we analyzed the expression of PST and STX transcripts in proteins immunoprecipitated from PC12 cells by anti -integrin mouse embryos using in situ hybridization. The results indicate that the antibodies indicates that PC12 cells express iS1 i3S and a amount of PST transcript is closely related to that of polysialic acid detected integrin subunits, and that exposure to Mn specifically a at 12 immunohistochemically, and both reach maximum embryonic day increases surface expression of the Alar subunit. In contrast, amount and acid declines (E12). The of PST transcript polysialic during arm is not isuunoprecipitated from a strain of PC12 cells that In a further development but they are still detectable at E15. contrast, is nonresponsive to the neurite-promoting effects of Mn. These when significant amount of STX transcript appears only in the later stages, data imply that Mn-induced neuronal differentiation of acid is reduced. These combined results PC12 polysialic already strongly suggest cells is mediated by integrins, in particular, integrins of the that the polysialylation of N-CAM during the early stages of development is This work to the a~rm subfamily. supported by a grant from the Howard mainly carried out by PST while both PST and STX contribute Hughes Medical Institute, Program for Undergraduate Science polysialylation in later stages of development. (Supported by CA33895) Education (#71191-523701).

740 741 ROLE OF CADHERINS DURING EARLY STAGES OF NEURULATION IN CHICK INHIBITION OF CELL MOVEMENTS DURING GASTRULATION BY EMBRYOS. (L.J. Marrero and5S.R. Hilfer) Biology Department, Temple University, MIS-EXPRESSION OF ZEBRAFISH OTX1 IN ZEBRAFISH EMBRYOS. Philadelphia, PA 19122) ((T. Murakami, O.G. Doerre, and E.S. Weinberg)) Department of Biology, University of Pennsylvania, Philadelphia, PA 19104. Neurulation, formation of the neural tube, has been shown to be driven by the coordination of a number of diverse morphogenetic cell behaviors. This research focuses We are studying the role of zOtxl, a zebrafish homeobox gene related to Drosophila on the role of two cadherins in earlier stages of neurulation in chick. Cadherins are a family orthodenticle, in the specification of the anterior brain during embryogenesis. zOtxl Ca2+ cell-cell adhesion. We have seen that of glycoproteins involved in the -dependent is expressed at mid-gastrula in a region that is similar or identical to the territory that antibodies two cadherins interfere with the normal of neurulation. Two against process fate maps to the future midbrain and forebrain. Interestingly, cells in this region do not antibodies against L-CAM (chicken E-cadherin) were used in this study; a polyclonal extensive movements characteristic of almost all other of the antibody against the Fab' fragments and a monoclonal antibody (7D6) which does not undergo epiboly parts When zOtxl is at levels the shown any activity in direct inhibition of cell aggregation. Both antibodies have the same gastrulating embryo. overexpressed high throughout effect, in which chick embryos cultured in the presence of the antibodies stopped or embryo by injection of zOtxl RNA at the 1-4 cell stage, embryos form defective delayed their growth as compared with control embryos. Scanning electron microscopy gastrulae which are characterized by aberrant epiboly and convergence movements, shows that the neural plate does not to undergo the normal shaping (that is, where the neural frequently resulting in a spina bifida phenotype. To trace individual cells that express plate thickens apicobasally, narrows transversely, and lengthens longitudinally). NCD-2, an zOtxl ectopically within the context of an otherwise normally gastrulating embryo, we antibody that blocks N-cadherin function does not interfere with the shaping of the neural co-injected RNAs encoding zOtxl and green fluorescent protein (GFP) into a single plate but rather interferes with a later process, the convergence of the folds. The folds are cell at the 16 cell stage. Descendents of the cell, recognized by GFP fluorescence, delayed to close in the presence of the antibody and in some cases the distance between the formed patches of clumped cells which did not undergo appropriate convergence folds increases. Fluorescent photographs show the patterns of expression of L-CAM in the movements and were eventually excluded from the body axis of the embryo. In ectoderm and N-cadherin at the neural folds. Confocal images are under reconstruction for a contrast, cells injected in this manner solely with GFP RNA, or with RNAs encoding three dimensional localization of the bound antibodies. These studies provide greater insight GFP and zOtx1 lacking a homeodomain, gave rise to descendents that scattered into the regulation of cadherin expression and function during the earliest stages of widely over the gastrula and eventually were located at many sites within the embryo. and the that cadherins can mediate cellular rearrangements neurulation support hypothesis These results indicate that expression of zOtxl can promote cell aggregation, and during tissue morphogenesis. (The authors are grateful to Dr. G. Grunwald and Dr. W. suggest that one of the roles of this protein during normal gastrulation may be to Gallin for providing some of the antibodies used in this project). create a territory of aggregated cells that eventually will define the prospective anterior brain of the embryo. 128a Neural Development I (742-747). Sunday

742 743 DEVELOPMENTAL EXPRESSION OF PST-1 AND STX, TWO EXPRESSION OF ACTIVIN A AND ACTIVIN RECEPTOR mRNAs DURING SIALYLTRANSFERASES THAT CATALYZE THE POLYSIALYLATION OF AVIAN NERVOUS SYSTEM DEVELOPMENT ((K. Kos and J. N. Coulombe)) NCAM. Phillips, L.A. Krushel, G.M. Edelman, and K.L. Crossin)) Department of Anatomy and Cell Biology, Uniformed Services University of the Health ((G.R. Sciences, Bethesda MD 20814 Department of Neurobiology, The Scripps Research Institute, La Jolla, CA Previous studies have suggested that the protein activin acts as a neurodifferentiation 92037. factor in the avian ciliary ganglion (CG). In the present study, we sought to determine whether activin and any of the known type activin receptor mRNAs were expressed in Polysialylation of the neural cell adhesion molecule (NCAM) has been shown chicken CG and dorsal root ganglia (DRG) during embryonic development to influence NCAM-mediated homophillic binding. A high level of PSA is In the CG, activin receptor type IIA (ActR type IIA) specific primers amplified a in the nervous during embryonic and early postnatal PCR product of the appropriate size for the type IIA receptor from all stages examined expressed system (E4-E18; stage 24- stage 45). Sequencing ofthe PCR productconftrmed that a fragment development and is thought to play a role in modulating cell-cell interactions of ActR type IIA was amplified. With in situ hybridization using ActR type IIA specific during cell migration, neurite outgrowth, and synaptogenesis. The gene digoxigenin-labeled riboprobes, we found that the ActR type IIA mRNA was restricted to encoding PST-1, a sialyltransferase that catalyzes the polysialylation of NCAM, cells with a neuronal morphology. Activin type IIB receptor mRNA was not detected. has recently been cloned from chinese hamster ovary cells (Eckhardt M. et al. Thus, activin signalling in CG neurons appears to be transduced by the ActR type Ilk Nature 373:715-718). In the present study, we have isolated rat PST-1 and a We also examined whether DRG neurons express mRNA forthe ActR type IA. ActR related sialyltransferase, rat STX, and have shown that both can sialylate NCAM type IIA specific primers used in rtPCR amplified an appropriate size PCR product in cDNAs from embryonic day 16 (El6) and post-hatch day 1 (Pt) chicken lumbar DRG. when transfected into the Neuro-2A neuroblastoma cell line. The expression Using in situ hybridization we found that only cells with a neuronal morphology were patterns of rat PST-1 and rat STX were examined by in situ hybridization using labeled with the ActR type HIA riboprobe. All of the neurons within the DRG hybridized probes specific for unique regions in each enzyme. PST-1 and STX were with the ActR type IIA riboprobes. Thus, like CG neurons, DRG neurons may have a expressed abundantly throughout the central and peripheral nervous system potential to respond to activin. and development and were co-expressed in most Activin A is thought to function as atarget-derived neurodifferentiation factor during prenatal early postnatal regulating the expression of somatostatin in neurons of the avian CGthat innervate the tissues examined. In the adult brain, PST-1 and STX were restricted to subsets choroid layer of the eye. In support of this hypothesis, we detected activin A mRNA in of cells within the olfactory bulb, cortex, hippocampus and cerebellum. In the microdissected choroid tissue by rtPCR. Interestingly, using rtPCR, we also found contrast to its prevalent expression during embryonic and early postnatal that activin A mRNAs is present in both CG and DRG. Which cell types express activin development, STX expression in the adult was reduced relative to that of PST-1. mRNA in these ganglia is not yet known. Our observations suggest that, in addition to These results suggest that polysialylation of NCAM is regulated by at least two activin's role as a target-derived noerodifferentiation factor in the CG, activin may play significant, and as yet unknown, roles in the development and function of DRGand other distinct sialyltransferases. We are currently exploring whether the two enzymes parts of the nervous system. (Supported by NSF tBN-9309932). function together to achieve the levels of polysialylation found in vivo.

744 745 SPATIOTEMPORAL PATTERN OF NP-1 EXPRESSION DURING XENOPUS EARLY DEVELOPMENTAL EVENTS IN THE PLACODAL COMPONENT OF DEVELOPMENT ((T. A. Moreno, M. Barembaum, and M. Bronner-Fraser)) AVIAN CRANIAL GANGLIA. ((J.W. Sechrist, M.R. Stark, B. Rowe, C. Marcelle, and Developmental and Cell Biology, University of California, Irvine, 92717 M. Bronner-Fraser)) Department of Developmental and Cell Biology, University of Califomia, Irvine, CA 92717. Neural crest cells are generated via interactions that occur between the neural plate and the epidermis. To examine genes potentially involved in The lack of markers to identify undifferentiated cranial placodes has been an neural crest formation, we recently isolated a gene called NP-1 from quail and obstacle in understanding the development of such structures as the trigeminal chick, by screening a day 2-4 chick library. NP-1 is expressed in the early ganglia. Application of four techniques in various novel combinations has added neural plate, and as development progresses, it becomes restricted to the new insights: 1) Dil surface labeling after neural tube closure followed by further neural folds, the dorsal region of the neural tube, and finally, to migrating incubation for up to 4 days reveals the precise distribution of all cranial placode neural crest cells as expression in the neural tube diminishes. Ablations of the derived cells at selected time neural folds in of as neural is intervals; 2) Double staining with neural markers such result up-regulation NP-1 expression crest as anti-neurofilament confirms that derived cells regenerated from the ablated side, suggesting that NP-1 may play a role in (NF-M) placode (DII labeled) are neural crest formation. Because it is difficult to test function in the chick neuroblasts; 3) Intense Pax3 gene expression overlaps the Dil labeled ophthalmic embryo, we have obtained the Xenopus homolog of NP-1 in order to study trigeminal placode and its migrated neuroblasts (Stark et al., this volume); 4) the function of the gene in a more accessible system. Xenopus NP-1 was Labeling the ophthalmic placode and its presumptive neurons with BrdU indicates cloned by screening a tailbud stage Xenopus library with the chick and some rostral neuroblasts cease DNA synthesis by the 10 somite stage even though mouse homologs as probes. A 1.9kb cDNA fragment with 94% homology to they do not exit the ectoderm until the 16 somite stage during ventral crest the chick and 90% homology to the mouse was isolated and used in whole migration. A few migrated placode cells apparently incorporate BrdU during a one mount in situ hybridization experiments. The expression of NP-1 is first hour pulse label but the majority are post-mitotic with the reverse true of placode detected at gastrulation, when the ectoderm is stained diffusely. Later at the ectoderm. Sensory nerve pathway formation can be initiated by pioneering axons neural plate stages, the expression domain narrows to include the neural (probably true of mandibular nerve) or by migrating placode derived neuroblasts plate and some of the flanking ectoderm. As development progresses, NP-1 differentiating as they move (a few unexpectedly forming the core of the proximal expression is detected in the dorsal neural tube, condensing cranial masses, ganglion as in the glossopharyngeal nerve); most intriguing is the finding that the neural crest, cranial ganglia, the otic vesicle, the eye, and the somitic ophthalmic nerve at the rostral midbrain level first extends axons within a small, mesoderm. In later stages, the brain is very strongly stained, as well as the longitudinal assemblage of neuroblasts derived from pre-pattemed overlying nuclei of the somites. The of expression pattern Xenopus NP-1 suggests ectoderm with the more posterior ophthalmic lobe proper and the axons it generates that it may play a role in early neural development, and functional studies to forming slightly later. The rostro-caudal (or disto-proximal) gradient in differentiation test possible roles for the gene in Xenopus are in progress. is consistent with a subtle equivalent gradient in neuroblast origin. These gradients may explain the presence of transient rostral ectopic ganglia In older normal embryos and displaced ganglia near the eye in experimentally manipulated embryos with heterotopic transplants or neural fold ablations.

746 747 COMPARISON OF CRANIAL AND TRUNK NEURAL CREST/TUBE USING GENERATION OF SMOOTH MUSCLE FROM TRUNK NEURAL DIFFERENTIAL DISPLAY. ((B.J. Martinsen and M. Bronner-Fraser)) CREST ((Kim Ward and M. S. Rao.)) Department of Neurobiology and Developmental and Cell Biology, Univ. of Calif., Irvine, CA 92717 Anatomy, University of Utah Medical School The avian neural crest is a useful model system in which to study cell fate Neural crest cells generate the peripheral nervous system as well as smooth specification because it gives rise to diverse cell types including autonomic muscle of the cardiac outflow tract, melanocytes and craniofacial connective neurons, sensory neurons, glia, pigment cells, endocrine cells, paracrine cells, tissue. We have examined the ability of trunk crest to generate cranial crest cartilage, and bone. Different populations of neural crest cells exist along the derivatives. We find that caudal neural crest cells can differentiate into cells rostrocaudal axis that form region-specific derivatives. For example, only that are smooth muscle actin (SMA) and desmin immunoreactive indicating cranial neural crest cells have the ability to develop into cartilage and bone. that they are smooth muscle cells. Desmin and SMA immnoreactive cells The factors that enable the neural crest to generate diversity among an initially arise from p75 immunoreactive crest cells that can also differentiate into homogeneous remain to be determined. One population possible mechanism neurons and glia. A transitional stage when occasional cells co-express SMA leading to fate specification could be that subpopulations of neural crest cells are pre-programmed to form specific cell types before the onset of migration. and p75 can also be identified. Smooth muscle differentiation can be In order to isolate molecules potentially involved in position-specific promoted by the addition of serum as well as identified molecules such as determination, chicken RNA isolated from cranial neural fold tissue (4-6 somite TGF-,O and retinoic acid. embryo) was compared to RNA from neural tube tissue (16-22 somite embryo) We have also identified a p75 immunoreactive immortalized neural crest using Differential Display. This technique used anchored and arbitrary primers cell line (MONC-1) derived from caudal crest that can generate smooth to generate cDNA fragments by reverse transcription followed by PCR. The muscle cells as well as generate neurons and glia. We show that smooth cDNA fragments were then electrophoretically separated and subsequently muscle differentiation can be enhanced by differing concentrations of retinoic compared. The differences in cDNA patterns indicated differences in the acid and to a lesser extent by TGF-13. PDGF however inhibits smooth mRNA levels and composition. The resulting 9 DNA sequencing gels were muscle differentiation. analyzed and 100 bands were kientified to be approximately only expressed in as a marker for we the cranial tissue and not in the trunk tissue. The Using p75 immunoreactivity migrating neural crest differentially displayed show that cardiac crest expresses to cDNAs were then isolated, sequenced, and used as probes for whole mount in p75 immunoreactivity prior migration, during migration and prior to desmin expression. Thus in vivo as in stku hybridization. One cDNA fragment, clone D9U6, only had expression in vitro, immunoreactive crest cells a the cranial neural folds from the most rostral region down to rhombomere 2. p75 represent precursor population that Another clone, D8U8, showed expression in migrating cranial neural crest cells. differentiate into smooth muscle cells of the cardiac outflow tract. The results verified that the isolated cDNAs were truly differentially expressed. Supported by a grant from the American Heart Association. Sunday. Neural Development I (748-751) 129a

748 749 PROSAPOSIN-INDUCED DIFFERENTIATION AND GANGLIOSIDE ARGININE KINASE IS EXPRESSED IN GROWING PROCESSES OF CNS EXPRESSION ON THE CELL SURFACE OF NS20Y NEUROBLASTOMA NEURONS AND MUSCLE PRECURSORS IN DEVELOPING DROSOPHILA CELLS. ((R.Misasi, M.Sorice, *G.S.Carson, T.Griggi, 'V.Dolo, 'A.Pavan, EMBRYOS. ((Y.E. Wang, P. Esbensen, and D. Bentley)) Division of G.M.Pontieri and *J.S.O'Brien.)) Dip. Medicina Sperimentale e Patologia, Neurobiology, Department of Molecular and Cell Biology, University of University di Roma "La Sapienza", Italy; 'Dip. Medicina Sperimentale, California, Berkeley, CA 94720-3200. University di L'Aquila, Italy; *Dept. of Neurosciences, University of California, San Diego Center for Molecular Genetics La Jolla, CA USA. Arginine kinase belongs to an evolutionarily conserved family of ATP- Prosaposin has been recently identified as a neurotrophic factor eliciting dif- guanidino phosphotransferases, whose members play an important role ferentiation in murine and human neuroblastoma cells (O'Brien et al., Proc. in energy metabolism. This enzyme exhibits significant sequence Nati. Acad. Sci. USA, 91,9593, 1994). In this study we analyse whether pros- identity to representative members of the guanidino kinase family, aposin and its active peptide (prosaptide) may modify the ganglioside pattern including creatine kinase. In cellular blastoderm (stage 5), arginine in NS20Y neuroblastoma cells. Cells were incubated for 24h in DMEM 0.5% kinase is expressed in the homogeneous cellular sheet surrounding the FCS containing prosaposin, 100 ng/ml. The analysis by high performance yolk. During gastrulation (stage 6-8), cells within the polar caps and thin layer chromatography did not reveal qualitative changes in the ganglio- midventral part of the blastoderm invaginate. These cells express high side pattern of prosaposin-treated NS20Y cells, compared to control cells, but levels of arginine kinase. Following gastrulation (stage 11), argK is the At stage it did reveal an increase of the content of all three major resorcinol positive expressed throughout neuroectoderm and the mesoderm. 12 and argK is restricted to the central nervous system: in bands (GM3, GM2, GD1a). The total ganglioside content, determined as beyond, neuroblasts and nascent neurons, and the visceral, somatic, and cephalic lipid-bound sialic acid, was 1.1 ± 0.24 mg/mg protein of untreated cells, 1.75 mesoderm: in muscle precursors and subsequently, muscle fibers. ± 0.36 mg/mg for prosaposin treated cells and 1.70 ± 0.30 mg/mg for prosap- tide treated cells. and immunofluorescence revealed Surprisingly, it is not expressed in ectodermal cells, PNS, or non- Cytofluorimetric analysis muscle mesoderm. This highly constrained expression pattern suggests that the increase of the ganglioside content was at the plasma membrane level. a specific role during embryogenesis. Arginine kinase is expressed The distribution in untreated and treated cells was an- ganglioside prosaptide throughout neurons: in the soma, the axon, and growth cones, but not in immunoelectron are in progress with the aim alyzed by microscopy. Studies the filopodia. Expression of argK in neurons is upregulated at the onset to on clarify whether the neurotrophic activity of prosaposin neuroblastoma of The in neurons of cell surface axongenesis. heavy expression growing and migrating cells might be mediated by the increase gangliosides. processes of muscle precursors suggests a role in outgrowth. (Supported by NSF #IBN 94-10065.)

750 751 IDENTIFICATION OF AN ENHANCER ELEMENT REGULATED BY DEVELOPMENT OF THE BASILAR AND AMPHIBIAN PAPILLAR BROAD-COMPLEX TRANSCRIPTION FACTORS IN THE CENTRAL NERVOUS BRANCHES OF THE XENOPUS EIGHTH NERVE. ((V. Lopez and E.E. SYSTEM OF DROSOPHILA. ((E. Liu, X. He and L.L. Restlfo)) ARL Division of Neurobiology, University of Arizona, Tucson, AZ 85721. (Spon. by E. Liu.) Serrano)), Biology Department., New Mexico State University, Las Cruces, New Mexico 88003. (Spon. by V. Lopez.) The Broad-Complex (BR-C), a steroid hormone-regulated gene which encodes a family of zinc-finger-containing transcription factors, sits near the top of a regulatory hierachy that controls metamorphosis in Drosophila We are interested in the ceHular interactions that take place during melanogaster. We have previously shown that the BR-C mediates specific auditory hair cell innervation and eighth nerve development. This study aspects of central nervous system (CNS) remodeling during examines the development of the axons of the auditory neurons of the metamorphosis, including assembly of visual system synaptic neuropils. In order to understand how the BR-C controls CNS re-organization, we are eighth cranial nerve in the African clawed frog, Xenopus aevws, during identifying BR-C target genes using an enhancer-detector method. We larval, juvenile and adult stages. Xenopus is a valuable model system screened a collection of autosomal P-element insertions by examining lacZ for these investigations, because like other amphibians, Xenopus absence reporter gene expression in the late larval CNS In the presence and inner ear lines of BRC transcription factors. One enhancer-etector strain, H217, shows a continuously produces sensory hair cells of the and lateral life. For this 52 68 I g striking alteration in 0-galactosidase (-gal) expresalon ppattern in the throughout its study, stage (larvae), stage (- absenceabof~BRC proteins.galactosidaIn wild-type C-5,H~a 217ectxpalressiondirects P-gal expression juvenile) and adult (2 40 g) animals were selected, and the eighth nerve in the medial optic lobes in cells we believe are neuroblasts of the Inner proliferative center. In B14-C null mutant CNS, p-gal expression is seen not was dissected, processed, embedded, and sectioned. I gtm sections only in the medial optic lobes, but also ectopically in the developing lamina, from each nerve branch were taken from a specific location in the two the synaptic target of retinal photoreceptors R1-6. Thus, BRC proteins auditory endorgans, the basilar papilla and amphibian papilla. Sections in the lamina, but in their normally repress B-gal expression developing from each were examined a Nikon TMD inverted absence, the H217-tagged enhancer is de-repressed.Ne have mapped the stage under Diaphot ectopic H217-directed expression pattern to the BR-C by using duplication microscope and analyzed for axon quantity and axon area using a JAVA and deletion chromosomes and three indepedently generated null alleles. computational image analysis system. Our data indicate that Furthermore, mutants of the brcomplementation group show the ectopic P- developmental increases in axon quantity are predominant between gal expression while those of the rbp and the 2Bc groups do not. Since the unctions represented by each of the mutant classes are thought to be larval and juvenile stages, while increases in axon area occur chiefly carried by different zinc-finger isoforms, we predict that repression of the between juvenile and adult stages. Future research will focus on SEM H217-tagged enhancer in the developing lamina requires BRC-Z2 proteins studies of the sensory neuroepithelia within each endorgan, and TEM but not -Z1 or -Z3 isoforms. Developmental studies suggest that the H217-tagged enhancer is activated by the rising levels of steroid hormones studies of myelin development in each nerve branch. This research was that trigger metamorphosis. Experiments are underway to characterize this supported by NIH (MBRS S06-GM08138-18; NIDCD R03DC01480-01) BRC-dependent enhancer genetically and molecularly. (Supported by NIH and Whitehall Foundation grants to EES. Grant NS28495) Axon Guidance (752-753)

752 753 AGRIN INHIBITS NEURITE OUTGROWTH BUT PROMOTES POSITIONAL MARKERS IN THE DEVELOPING RAT SPINAL CORD ATTACHMENT OF EMBRYONIC MOTOR AND SENSORY NEURONS ((D. Kapitula, Q. Zhu, R. Imondi, Z. Kaprielian.)) Department of IN VITRO. ((S. Woo, D. Chang, J. Campanelli*, M.J. Ignatius.)) Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 Department of Molecular and Cell Biology, University of California, Region-specific cell surface molecules are thought to play important roles in Berkeley, Berkeley CA 94720; *Department of Biochemistry, University of pattern formation and axon guidance in the developing vertebrate central ner- Illinois, Urbana, Urbana, IL 61801 vous system (CNS). The cyclophosphamide immunosuppression method was Agrin is a secreted glycoprotein with the ability to cluster cell surface used to generate monoclonal antibodies (mAbs) that recognize antigens which molecules, including the nicotinic acetylcholine receptor on muscle cells. Al- are expressed at high levels in retinoic acid (RA) treated embryonal carcinoma ternative splicing of agrin mRNA produces a family of four isoforms that differ cells (NT2/Dl), and label the developing CNS in a regionally restricted man- in their clustering ability. Neuronal-specific isoforms with high clustering abil- ner. mAbs CARO 2 and CARO 5 (for CAudalROstral) preferentially label ity play a role in AChR aggregation at the neuromuscular junction. However, the floor plate in E12 E18 rat embryos, and bind the surfaces of NT2/D1 non-neuronal agrin expressed in many tissues exhibit low clustering ability and and dissociated embryonic rat spinal cord cells. RA upregulates the expres- have no known function. Monolayer cultures of CHO cells expressing either a sion of the corresponding antigens. Immunoblotting of the detergent soluble neuronal (agrin-19) or non-neuronal (agrin-0) form of chick agrin, with drug- membrane fractions derived from NT2/D1 cells or embryonic rat spinal cord, resistant controls, were used to assay the effect of agrin on neurite outgrowth shows that both mAbs recognize a single 27 kD protein. Hoever, mAb CARO and cell attachment. When axons of either sensory cells or two populations 2 immunoprecipitates a 60 kD doublet from NT2/D1 cells, while mAb CARO of motor neurons confronted agrin-expressing CHO cells, their growth was ar- 5 immunopecipitates a 20 kD protein from both NT2/D1 and E16 rat spinal rested. Neurite extension on confluent monolayers of agrin-expressing CHO cord cell extracts. Immuno-screening of a NT2/D1 bacterial expression cDNA cells was also substantially reduced below that of controls. Time course ex- library using mAbs CARO 2 and CARO 5 has identified eight positive clones. periments suggest that neurite outgrowth is stopped and not simply retarded. The sequence information obtained thus far has failed to identify the CARO Attachment of neurons was nearly two-fold higher to agrin monolayers than 2 and CARO 5 antigens as known proteins. These findings suggest that the to control cells, showing that inhibition is not a result of a non-permissive en- CARO 2 and CARO 5 cell surface antigens are novel floor plate markers that vironment. These results suggest additional roles for agrin in synaptogenesis: may play a role in neural tube patterning and/or axon guidance. Supported agrin may function as a stop signal at the myofiber surface and may anchor by Research Grant #95-17 from the Whitehall Foundation. the pre-synaptic fibers to the eventual motor endplate.