Monday. and 11 (936-941) 161a

936 937 CHL4IMIDOAIONAS FLAGELLAR DYNEIN IS ACTIVATED BY INHIBITORS OF THE 'DYNEIN REGULATORY COMPLEX' RESIDES ATTHE JUNCTION BETWEEN CALMODULIN AND cAMP DEPENDENT KINASE. ((D.R Howard, E.F. Smithl, G. THE RADIAL SPOKES AND THE INNER DYNEIN ARMS. ((E.T. OToole, L.C. Habermacher, and W.S. Sale)) Dept. of Anat and Biology, Emory University. Gardner, T.H. Giddings, and M.E. Porter) Dept. of Molecular, Cellular, and Atlanta, GA 30322. tDept. of Genetics and Cell Biology, Univ of Minnesota, St. Paul, MN. 55108. Developmental Biology, University of Colorado, Boulder CO 80309 and Dept. of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455 We have used a quantitative measure of dynein-driven sliding velocity and mutant strains of Chlanssydomoonas to demonstrate that flagellar dynein activity is Previous stucies in Chlamydomonas reinhardfli have identified six axonemal regulated by the radial spokes via a stable modification (Smith and Sale, 1992, Science. polypeptides as components of a 'dynein regulatory complex' (DRC). This complex 257:1557). One hypothesis to explain these results is that a dynein component(s) is is thought to coordinate the activity ofthe multiple flagellar (Huang et a/., Cell regulated by a post-translational modification such as phosphorylation/dephosphorylation. 28:115-124, 1982; Piperno et al., JCB We have used We have used a pharmacological approach to test this hypothesis. Treatment of 118:1455-1463, 1992). Chlamtydonionas cells deficient for radial spokes (pJ14, pJl4pj28) with calmodulin improved FPLC procedures (Kagami and Kamiya, J. Cell Science 103:653-664,1992) antagonists (TFP, W-7, calmidazolium) increases the velocity of dynein-driven and computer averaging of EM images (Mastronarde et a/., JCB 118:1145-1162, microtubule sliding to wild type values. Treatment ofp.14 cells with inhibitors (H-8, HA- 1992) to probethe physical relationship between the DRC and the dynein arms. Our 1004) of cAMP dependent kinase (PKA) also increases dynein-driven microtubule results suggest that the DRC components are located at the base ofthe second radial sliding velocity to wild type values. Inhibitors of calmodulin and PKA have no effect on spoke in close association with the inner dynein arms. (1) Averages of axoneme the microtubule sliding velocities of control, spoke-containing axonemes (137c, In pj28). cross-sections indicate that a mass in the inner arm region is significantly reduced in vitro phosphorylation of axonemes reveals that axonemes from spokeless mutants incorporate three to five times more phosphate than spoke-containing axonemes. three DRC mutants (pf3

938 939 A 29 kDa cAMP-DEPENDENT PHOSPHOPROT'EIN IS DIRECTLY SEA URCHIN PROTEIN PHOSPHATASES DEMONSTRATE SUBSTRATE ASSOCIATED WITH AND REGULATES AXONEMAL 22S DYNEIN. ((K. SPECIFICITY WITH RESPECT TO INNER AND OUTER ARM DYNEINS ((J.S. Tash Barkalow, T. Hamasaki, S. Nair and P. Satir)) Department of Anatomy and and G. E. Bracho)) Department of Physiology, University of Kansas Medical Center, Structural Biology, Albert Einstein College of Medicine, Bronx, N.Y. 10461. Kansas City, KS 66160

A 29 kDa phosphoprotein (pp29) that copurifies with 22S dynein of Protein phosphorylation is a major pathway for initiation and modulation of sperm Parameciun tetraurelia is phosphorylated or thiophosphorylated in a cAMP- motility. We have recently purified and characterized a unique 23Kd protein phospha- dependent, Ca2+-sensitive manner. This phosphorylation regulates the speed tase (23K-PPase) that was released from sea urchin sperm by homogenization. In ofmicrotubuletranslocation by 22S dynein in vitro. After thiophosphorylation addition, bound PPases were identified in isolated flagella. The bound PPases were within the axoneme, pp29 has been isolated away from the heavy chains and characterized by their sedimentation pantern on sucrose gradients relative to 21S outer partially purified. Purified 22S dynein not otherwise treated retains sub- arm dynein (OAD) and the 11S and 21S regions of IAD (inner arm dynein). The isola- stoichiometric amounts of pp29. In a reconstitution assay the partially purified ted PPases were tested for their ability to dephosphorylate cAMP-dependent phospho- pp29 specifically binds to purified 22S dynein, but not to 14S dynein (a single cosedimenting with OAD and lADs, and effects upon microtubule gliding in headed molecule) nor BSA. Specific association with 22S dynein can be vitro. In OAD extracts, a broad peak of PPase activity sedimented near the top of the competed away by using a cold pp29 fraction that has or has not been gradient. No activity was found in the fractions containing 21S OAD. In IAD extracts, experimentally thiophosphorylated. An in vitro microtubule motility assay was PPase actvity was present 1) as a major peak near the top of the gradient, 2) an activi- used to test the effect of rebinding. When 22S dynein with rebound functional ty that included the 11S region, and 3) at the bottom of the gradient, distinct from the pp29 was used, microtubule translocation velocity increased over controls. 21S region. 21S OAD contained cAMP-dependent phosphoproteins corresponding to Velocity increased further when rebound pp29 was thiophosphorylated, the a-heavy chain (HC) and a 23Kd subunit. There was no phosphorylation of OAD by suggesting that the reassociation is functional. Paramecium 22S dynein can be protein kinase C. The 23Kd OAD subunit was dephosphorylated by the OAD proteolytically digested with chymotrypsin to yield one-headed and two- PPase, the IAD PPase from the bottom of the gradient and the 23K-PPase. The OAD HC was headed structures. The pp29 binds preferentially to the one-headed fraction. dephosphorylated by the OAD PPase and 11S IAD PPase. 21S IAD contained Based on these results pp29 is probably a regulatory light chain (DLCr) of a region specific heavy chain of 22S dynein. When the pp29 fraction from Paramecium a major phosphoprotein at 15OKd which was not dephosphorylated by any of the PPas- es. The S IAD HC, 150,54 is mixed with Tetrahymena 22S or 14S dynein, pp29 is capable of binding to 11 contained and 39Kd phosphoproteins as well as endoge- nous PPase activity. In S only Tetrahymena 22S but not 14S dynein. This suggests that the role of pp29 is 11 IAD, the 54Kd phosphoprotein was desphosphorylated conserved at least within ciliates. In that other organisms have similar cAMP- by the 23K-PPase. Dephosphorylation of the OAD 23Kd subunit was associated with induced responses, a pp29 homolog may be operative in their cilia. Amnino a diminished gliding velocity of in vitro. These results suggest that distinct acid sequences from isolated pp29 have been obtained. We are analyzing PCR sperm PPase activities may be selective regulators of the function of the different forms products derived using oligonucleotides based on these sequences. of flagellardynein. Supported by NIH-GM29496 (JST) and the Andrew W. Mellon Foun- dation (JST) and NIH-HDO2528 (KUMC).

940 941 EXPRESSION OF A CYTOPLASMIC DYNEIN HEAVY CHAIN CYTOPLASMIC DYNEIN AND GLUED COLOCALIZE THROUGHOUT EARLY GENE IN DIROSOHILA ((M.-G. Li, M. Serr and T. Hays)) Dept. of DROSOPHILA DEVELOPMENT (( M. McGral, A. Siivanovich, and T. Hays.)) Genetics and Cell Biology, University ofMinnesota, St. Paul, MN 55108 Dept. of Genetics and Ce- Biology University of Minnesota, St. Paul, MN 55108

Microtuble motors may play an important Cytoplasmic dynein is amicrotubule-associated motor protein complex. Itis role in a number of microtubule- dependent processes In Drosophila composed of two heavy chains and multiple intermediate and light chains. oogenesis and early development. Of particular interest is the hypothesis that a matemally expressed cytoplasmic are energy-transducing ATPases that move their associated cargoes Dyneins dynein participates in the determination of the and In the poskioning of towards the minus end ofmicrotubules. We have taken amolecular genetic anterior and posterior determinants wihin theoocyte. In the eariy embryo, the approach to understand the function of cytoplasmic dynein. In previous work matemally supplied dyneln motor may also be utilized during nuclear division and we have isolated the gene Dhc64C which encodes a cytoplasmnic dynein heavy migration. We have previously cloned and sequenced a cytoplasmic dynein chain in Drosophila- The gene is transcribed into a 14.3kb mRNA that encodes heavy chain gene (Dhc64C) thatis abundantly expressed In embryos (Li, et al., a protein of 4639 amino acids. To investigate the developmental processes in submitted). In Drosophila, the Glued locus encodes a homolog of the 150 kD which the Drosophila dynein Dhc64C participates, we have characterized the subunit of (Holzbauret al.,1991), an activator of dynein-medlated vesicle expression of the gene. Northern blot analyses show this gene is expressed in motility (Gill et al., 1991). The Gluedgene has also previously been cloned and all tissues examined, and throughout embryogenesis. To further examine the sequenced (Swaroop, et al., 1987). We have raised antibodies against spatial distribution of Dhc64C transcripts dunng embryogenesis and larval encoded by both Dhc64C and GAvedcDNAs. We have examined the spatial and temporal distributlon of dynein and Glued throughout and development, we engineered lacZ constructs that are driven by gene oogenesis early the embryogenesis using immunocytochemical methods. Our results show that the These constructs were reintroduced promoter. reporter into Drosophila by P two polypeptides co-localize throughout early development. Dynein and Glued elerent-mediatedtrnsformation and the expression of the reporter was are expressed very earlyin oogenesis and become enriched in one of sixteen monitored in sint Heavy lacZ staining is found in the testis and all stages of cells that will develop as the oocyte. By mid-oogenesis, a subcellular embryogenesis. Staining is also detected throughout oogenesis, in both nurse concentration of both dynein and Glued Is observed at the posterior end of the cells and . In larva, intense staining is seen in the centrl line of ventral oocyte. Studies using microtubule inhibitors indicate that the localization of ganglion of brain and in all imaginal discs. Other larval tissues such as dynein to the posterior of the oocyte is microtubule-dependent. Posterior intestines are unstained or weakly stained. Our analysis of the expression of localization of dynein and GluedIs coincident with that of the staufen gene the Dhc64C gene indicates that the gene is abundently expressedthroughout product, which is known to be required for the positioning of anterior and to co- development and is most likely an essential gene. Genetic screens to isolate posterior determinants. In the early embryo, both dynein and Glued appear localize with microtubules of the cell lethal mutations in the heavy chain gene are underway. (This work is throughout all stages cycle, including supported by NIH, MOD, and the PEW Charitable Trust.) mitosis. Mutations are being exploited to determine the function of dynein and GluedIn Drosophila oogenesis and embryogenesis. This work Is supported by NIH, ACS, and the PEW Charitable Trust. 162a Dynein and Kinesin II (942-946). Monday

942 943 ANALYSIS OF THE p1500GUed-CENTRACTIN COMPLEX REVEALS A CHARACTERIZATION OF THE HUMAN cDNA ENCODING pl50GLUED MICROTUBULE BINDING FUNCTION IN VIVO AND IN VITRO. ((C. M.Watemian- AND ITS EXPRESSION IN THE HUMAN NTERA 2 CELL LINE. ((M. K. Storer, S. Karki and E. L.F. Holzbaur)) University of Pennsylvania School of Veterinary Tokito, V. M.-Y. Lee*, and E. L. F. Holzbaur)) University of Pennsylvania Medicine, Philadelphia, PA. School of Veterinary Medicine, and *Univasity of Pennsylvania School of Medicine, Philadelphia. PA 19104-6046. p15OGkuedwas first identified as a polypeptide which copurities with microtubules (MTs) along with the minus end-directed MT-based motor, cytoplasmic dynein. pl as a with p5OGluedhasbeen shown to exist as a member of a muruisubunit complex with OGlued, originally identified polypeptide co-purifying cyto- of is a component a polypeptides 50 and 62 kDa, as well as with the novel , centractin (42kDa). In plasmic dynein, of distinct 20S cellular complex which also spite of its apparent biochemical association with MTs, it was uncertain whether contains the novel form of actin, centractin. The molecular analysis of the 150 kD rat pl5OGiuedbinds MTs directly or via secondary protein interactions. This uncertainty cytoplasmic dynein-associated polypeptide from revealed homology with was reflected in vivo by the various, yet specific imrrunolocalization pattems that the Drosophila Glued] mutation, which is characterized by defects in neuronal plSOGlUeddisplayed in cultured cells, including a punctate cytoplasmic localization in development in the heterozygote, and is lethal when homozygous. We have log growth cultures, the centrosome in confluent cultures, kinetochores during now isolated and characterized the homologous cDNA from a human fetal brain prometaphase and spindle fibers during metaphase. Recently, was shown that library. Overall, the human cDNA clone is -80% homologous to the rat cDNA amino acids 26-81 of pi5OGlUedhad extensive homology to a putative MT binding sequence. Comparisons of the predicted polypeptide sequences of the human motif characterized in CLIP-170, a protein implicated in linking endocytic vesicles to and rat clones have allowed the identification of highly conserved regions MTs. In order to examine the function of pl5OGlued in vivo, we have subcloned the within the 150 kD polypeptide including sequences with homology to rat brain cDNA encoding pl50Gluedinto an eukaryotic expression vector for cytoskeletal binding motifs. Biochemical data from our laboratory support the overexpression in Rat-2 fibroblasts. Transient overexpression of the full length cDNA hypothesis that these motifs enable the 150 kD polypeptide to directly cross-link (1325 amino acids) and C-terminal deletion constructs of A243, A383, and A514 microtubules to centractin, thus providing an interaction between the amino acids, respectively, all result in similar immunofluorescence localizations. In microtubule-based and actin-based cellular . We have also transfected cells, the overexpressed protein constructs decorate the entire length of examined the expression of pl5OGlUed in NT2-N cells, a human interphase MTs as shown by co-localization with anti - antibodies. This in vivo teratocarcinoma cell line induced to differentiate to a stable neuronal evidence for MT association is corroborated by in vitro MT binding assays. The phenotype full by retinoic acid. is expressed the axonal of these length pSOGkuedand C-terminal deletion constructs translated in vitro in rabbit plSOGlued along projections consistent with a in reticulocyte lysates all demonstrate salt-dependent cosedimentation with MTs cells, role for the polypeptide retrograde microtubule-based vesicular axonal transport. In differentiated NT2-N cells we have found that assembled from purified bovine brain tubulin. These results suggest that pi5oG/ued contains an N-terminal MT binding domain which may mediate the interaction between multiple isoforms ofpI5OGlued are expressed which differ in their the pl500GIedcomplex, cytoplasmic dynein, and the microtubule cytoskelelon. microtubule-binding characteristics, and which may represent alternatively (Supported by NIH GM48661) spliced forms which we are currently characterizing. Supported by the MDA.

944 945 TRANSFECTION OFCOS-7 CELLS WITH CYTOPLASMIC DYNEIN INTERMEDIATE CLONAL ANALYSIS OF KINESIN FUNCTION IN DROSOPHILA. CHAINS. ((K.T. Vaughan and R.B. Vallee)) Cell Biology Group, Worcester ((R.P. Brendza, K.B. Sheehan and W. M. Saxton)) Department of Biology, Foundation for Experimental Biology, Shrewsbury, MA, 01545 Indiana University, Bloomington, IN 47405.

Cytoplasmic dynein 105:contains a complex set of accessory subunits (Paschal at Kinesin is a microtubule-associated motor protein that utilizes chemical energy al., 1987, J. Cell Biol. 1273-1282), whose function in retrograde transport derived from the hydrolysis of ATP to generate the force necessary to travel and mitosis are poorly understood. The recent cloning of the rat cytoplasmic along microtubules toward plus ends. In D.melanogaster, homozygous dynein 74kD intermediate chain(Paschal eta/., 1992, J. CellBiol.1l_8: 1 133- mutations in the kinesin heavy chain gene (khc) result in lethality during the 1143) revealed homology with the Chiamydomonas axonemal dynein second or third larval instar. The pre-lethal phenotype of khc mutant larvae intermediate chains making the 74kD subunit of cytoplasmic dynein a likely indicates that kinesin is essential for motor funcdon. Specific neuronal candidate for direct binding to organelles and kinetochores. As a first step in defects include the inhibition of action potendal propagation and impaired elucidating the role of this polypeptide, we have examined its distribution by release. To identify non-neuronal celltypes that require kinesini both conventional immunocytochemistry and by expression ofepitope-tagged function, we are generating and analyzing genetic mosaics in different tissues. A FLP system was to constructs in COS-7 cells. Polyclonal antisera were prepared against the wild- recombinase/FRT mitotic recombination utilized generate are type 74kD protein expressed in E. co/i and purified by nickel-affinity cells that homozygous for a severe khc mutation in otherwise heterozygous chromatography. The antisera primarily label small, detergent-extractable first and second instar larvae. Homozygous khc mutant stem cells were capable of eye tissue. vesicular structures concentrated in the juxtanuclear region of cells surrounding founding clonal patches of adult Judging from the largest clone size (90 ommatidia), an eye stem cell can proliferate through at least11 the Golgi apparatus but extending at lower density to the cell periphery. Staining division cycles after loss of the wild-type khc gene. A search for subtle defects is in was excluded from the nucleus during interphase, suggesting that the nuclear progress via comparison of the division rates and of the ultrastructure of mutant localization signals encoded in the 74kD polypeptide are nonfunctional. In and wild-type cells. To determine if kinesin function is required in transient transfections using 74kD constructs myc-tagged at either the amino oogenesis, or germline stem cells that are homozygous for a severe khc mutation have carboxy terminus, a more intense but comparable distribution was been observed, generated using X-ray induced mitotic recombination in third instar larvae. Such though a detectable background of soluble protein was also seen. This khc mutant stem cells can found egg-producing ovarioles, but the eggs appear observation suggests that potential binding sites are not saturated by morphologically abnormal and fail to enter embryogenesis. Control eggs endogenous cytoplasmic dynein during interphase, and that the myc-tag does produced by analogous recombination events but containing a wild-type khc gele not adversely affect the structure of the expressed polypeptide. These develop into normal larvae and adults. From these and other data, we believe that constructs are being to used to study post-translational modification of kinesin function is required in oogenesis. Experiments to identify specific cytoplasmic dynein subunits as well as the effects of mutant forms on function. defects in oogenesis caused by impaired kinesin function are in progress. (This work was supported by NIH grant GM1 5941 and the MDAIKTVI and NIH grant GM47434(RBVI).

946 NEURITE OUTGROWTH IN PC12 CELLS IS INHIBITED BY ANTISENSE OLIGONUCLEOTIDES TO A MURINE KINESIN HEAVY CHAIN ((L.P. Zhao, J.S. Koslovsky, C.E. Stewart, and J.A. Mercer)) Department of Physiology, University of Texas Southwestem Medical Center, Dallas TX 75235-9111.

We have isolated a cDNA representing the motor domain of a kinesin heavy chain of the unc-104 family (KHCA) by screening a mouse brain cDNA library with a PCR product of degenerate primers representing conserved sequences in the motor domain. This cDNA is similar, but not identical, to KIF1 (Aizawa et aL., JCB 119:1287). KHCA transcripts of approximately 11 and 8 kb are expressed at high levels in brain and skin. In situ hybridization analysis showed that KHCA is expressed at highest levels in the hippocampus and cerebellar nuclei of the aduit brain. We have used both normal PC12 cells and PC12 cells overexpressing trk that exhibit very rapid neurite outgrowth for antisense inhibition studies. Three different antisense oligonucleotides inhibited neurite outgrowth at concentrations of 20-50 lM, while the corresponding sense oligonucleotides at the same concentration had no effect. The number of neurites per cell was unaffected; the average neurite length was 7.5FM in sense controls and 3.3FM in antisense-treated cells. These data suggest an important role for this kinesin in neurite outgrowth. Monday. Microtubule Dynamics and Assembly II (947-952) 163a

947 948 REGULATION OF MICROTUBULE DYNAMICS BY THE SEPARATION OF YEAST oa- AND J-TUBULIN UNDER NON- CONTROLLED HYDROLYSIS OF A GTP ANALOGUE AND DENATURING CONDITIONS ((V. Praitis, B. Weinstein, and F. Solomon)) EVIDENCE FOR A GAP IN XENOPUS EGG EXTRACT THAT IS Department of Biology and Center for Cancer Research, Massachusetts DIRECTED AT MICROTUBULE-GTP. ((M. Caplowv. J. Shanks and Institute of Technology, Cambridge, MA 02139 R. Ruhicn) ) Dcpartment of Biochemistry, University of North Carolina, Chapel Hill, NC 27599 We are interested in studying the properties of the a- and B-tubulin polypeptides and how these relate to in vivo function. We report here the separation of a- and 8-tubulin monomers under non-denaturing conditions. We have detcrmined the effect of nucleotide triphosphate hlydrolysis on Both a- and B-tubulin from yeast soluble protein extracts in Pipes buffer can microtubule dynamics using microtubules containing a GTP alialogLic be inmmunoprecipitated by the anti- antibodies A I BG7 and B lBE2, (GMPCPP), in which a methylene moiety links the alphla and beta generated against a- and 13-tubulin, respectively. This association is stable; phosphates. Usling a video microscopy assay the rate of tubulin- repeated exposure to additional buffer fails to elute either polypeptide. This GMPCPP subunit dissociation wvas found to be 0.6-1.6 sec'1; this is behavior is consistent with previous evidence that the tubulin dimer has a about 1000-fold sloNvcr than Nvith tubulin-GDP subunits. Althotighi the weak dissociation constant. However, the addition of a small amount of non- half-life for hydrolysis of GMPCPP in microtubules is about 10 hours, ionic detergent to the immunoprecipitated tubulin solubilizes that tubulin chain not the For in the the analogue was 87% hydrolyzcd in 10 min in 60% glycerol. After specifically recognized by antibody. example, glycerol-indticed GMPCPP hydrolysis, tubulin-GMPCPP subunits presence of detergent, virtually all the a-tubulin remains associated with the A1BG7 antibody, while as much as 85% of the B-tubulin is eluted. Similarly, dissociated at a ratc of approximately 136 sec- enid) anid 32 I(pltis as much as 67% of a-tubulin elutes from B 1BE2 immunoprecipitates. The secc I (mlitis enid) The greater subunit dissociation rate after hydrolysis detergent-mediated separation of 13-tubulin from s-tubulin in A1BG7 proves can that microtubulc dynamics be regulated by nucleotide experiments is significantly inhibited by the inclusion of 1 mM GTP. As hvdrolvsis. GMPCPP in microtubules was found to be rapidly much as 95% of the 8-tubulin remains associated with AIBG7 antibody. hydrolyzed anid GMPCPP-inicrotubules are disassemilbled in anl extract These data suggest that the addition of GTP stabilizes the dimer. We have fromn activated Xenopus eggs, in a reaction that wvas stimulated by evidence that the separation of tubulin dimer is not due to irreversible cyclin A 90. Tllis appears to rcsult from action of a microtubule denaturation. First, the concentration of non-ionic detergent does not inhibit GTPase activating protein (GAP) which may regulate the microtubuile microtubule polymerization. In addition, in the absence of detergent, eluted catastrophie frequency in cells. Supported by grant GM46773. c-tubulin can be immunoprecipitated by B 1BE2 antibody, but only in the presence of B-tubulin, suggesting that the separate chains can reassociate.

949 950 LOW DOSE EFFECTS OF TAXOL ON MICROTUBULES AND LOW CONCENTRATIONS OF TAXOL SUPPRESS MICROTUBULE CYTOSKELETAL ORGANIZATION. and L. DYNAMICS DIRENTIALLY AT PLUS AND MINUS ENDS BY ((S. Lee, SUBSTOICHIOMETRIC BINDING TO HIGH AFFINITY SITES Armstrong)) Department of Tumor Biology, Schering-Plough ((W. B. Denfy, L. Wilson, & M. A. Jordan)) DepL Biol. Sciences, Research Institute, Kenilworth, NJ 07033 University of California, Santa Barbara, CA 93106. The mechanism by which taxol promotes the assembly of Taxol, at low concentrations, blocks mitosis in HeLa cells without enhancing the levels of polymerized microtubules (MTs), apparently by stabilizing spindle microtubules remains largely unknown. We used MT dynamics (Jordan et al, PNAS, in press). We are investigating the immunofluorescence microscopy to study the effects of mechanisms by which taxol supprsses growing and shortening dynamics at MT taxol on cytoskeletal organization in Human Umbilical Vein ends (by video microscopy) in relation to the binding of [3H]taxol to the MTs. Endothelial Cells (HUVECs). Our results demonstrate that Taxol (10 nM to 20 pM) was added to steady-state MTs assembled with bovine At below 100 taxol concentrations less than luM bind preferably to the brain tubulin depleted of associated proteins. concentrations nM, taxol had no effect on the critical concentration, but suppressed dynamics in microtubules (MTs) and not to tubulin dimers. These taxol- association with the binding of very small quantities of taol to the MTs. For stabelized MTs are resistant to depolymerization by cold example, at 50 nM taxol, 1 molecule of taxol was bound per 417 molecules of treatment and show increased flexibility in a time- and tubulin in MTs. At this concentaon, the plus end growing rate was but the rate was inhibited 55%. Rates of and manner. At unaffected, shortening by growing concentration-dependent concentrations shortening at the minus ends were unaffected, but the minus ends spent a higher greater than luM, taxol promotes the assembly of new percentage of time in a state of undcable (attenuated) activity in the presence near the nucleus but not near the cell periphery. of taxol (49%) than in its absence (37% ). At 100 nM taxol and above, the These taxol-assembled MTs form a dense network which ratios of bound taxol to total tubulin in MTs increased to approximate stoichiometric levels and the critical subunit concentration was reduced. replaces the normal MT organization, and excludes Growing and shortening rates were significantly inhibited at both ends and the (MF) structures. However, the MFs minus ends became indistinguishable idnetically from plus ends. Based on reorganize to occupy the same area as the MTs and the binding studies with [3H]taxol, there appear to be two different affinity classes cytoplasmic membrane retracts to surround the of taxol binding sites on Mrs. Substoichiometric binding of taxol to high data that MTs determine affinity sites may be responsible for the suppression of dynamics and inhibition . These suggest may of mitosis at low taxol concentrations. Supported by NIH NCI CA57291. cell shape, cell size and cytoskeletal organization.

951 952 CHARACTERIZATION OF HELA CEIL TUBULIN MICROTUBULE MICROTUBULE DYNAMICS IN FISH MELANOPHORES STUDIED BY DYNAMICS IN VITRO ((D. A. Thrower, M. A. Jordan, and L. Wilson)) PHOTOACTIVATION OF CAGED FLUORESCEIN LABELED TUBULIN. Dept. Biol Sci., Univ. California Santa Barbara, CA 93106. ((V.I.Rodionov and G.G.Borisy)) Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53705, and A.N.Belozersky Institute of Physico- Most in vitro studies on microtubule (MI) growing and shortening dynamics Chemical Biology, Moscow State University, Moscow 119899, Russia. (dynamic instability) have utilized brain which are derived from a heterogeneous and largely non-dividing cell population. We have begun to Microtubules in fish melanophores form a regular radial array which is known characteize the dynamics of MTs composed of tubulin (purified by to be important for centripetal and centrifugal movement of melanosomes. Our tpatr e-dependent assembly and disassembly and phosphocelluose previous experiments with the use of injections of X-Rhodamine labeled tubulin chromatography) from HeLa cells, a rapidly-growing human tumor cell line. showed that microtubules in melanophores exchanged rapidly tubulin subunits We find that the growing and shortening dynamics of the HeLa cell MTs are with tubulin from the soluble pool. To further characterize and quantitate significantly reduced compared with MTs assembled from bovine brain tubulin dynamic properties of melanophore microtubules we now have used tubulin purified and analyzed by the same conditions. The three most significant labeled with caged fluorescein. Modified tubulin incorporated into microtubule differences found thus far between HeLa cell Mrs and brain MTs (each network and fluorescent bar could be induced by irradiation of cells with polymerized at 1 mg/ml) respectively are; growing rate: 1.6 ± 0.1 mi/mn and aggregated pigment. Moreover, the same technique was found to be applicable to cells with dispersed pigment as photoactivation of caged fluorescein by 3.9 ± 0.5 pm/min; shortening rate: 1.5 ± 0.1 pm/mi and 8.8 ± 1.8 gm/min; irradiation at 360 nm did not induce cell damage. In both melanophores with and percent of dme spent in a phase of attenuation (no measurable length aggregated and dispersed pigment, the fluorescent bar did not move relative to change): 57 ± 4%, and 15 ± 2%. Further purification ofHeLa cell tubulin by the cell center and disappeared within one hour. Quantitative analysis of the FPLC (MonoQ resin) to remove faint bands present on silver-stained dissipation of fluorescence after photoactivation was consistent with the results polyacrylamide gels does not alter the dynamic parameters. However, addition of previous experiments and confinmed the dynamic nature of melanophore of HeLa cell cytoplasmic exats depleted of tubulin results in increased microtubules. As early EM studies suggest microtubule depolymenization during dynamic instability of the HeLa cell Mrs. Crowing and shortening rates of pigment aggregation it is of special interest whether microtubule dynamics HeLa cell MTs in the presence of the cytoplasmic extract are increased to 3.9 change accompanies pigment motion cycles. Experiments are in progress now 0.1 pn/min and 4.5 0.2 pm/min respectively, and the percent time in which will help to elucidate whether microtubule dynamics in fact changes attnuation is reduced to 20 5%6. The results suggest thiat tubulin from some during melanosome aggregation and dispersion. Supported by NIH grant dividing cells forms Mrs that are intrinsically less dynamically unstable than GM25062 to GGB. Mrs assembled with neural tubulin and that the MIs can become more dynamically unstable trugh the acdon of intacellular regulatory factors. Supported by ACS DHP-43E. 164a Microtubule Dynamics and Assembly II (953-958). Monday

953 954 STUDY OF FACTORS THAT AFFECT THE RATE OF GTP HYDROLYSIS GMPCPP ENHANCES NUCLEATION AND INDUCES COLD ON STABILIED MICROTUBULES ((L. D. Belmont and T. J. STABLE RIBBONS AND MICROTUBULES. ((B.I. Vulevic*, J. Mitchison)) University of California at San Francisco, Dept. of Turner* and J.J. Correiar)) *Dept. of Biochemistry, Univ. of Biochemistry and Biophysics, San Francisco, CA 94143. Mississippi Medical Center, Jackson, MS. 39216. In an attempt to further understand the regulation of We have synthesized the GTP analog GMPCPP according to the microtubule (MT) dynamics, we have developed an assay look at method of Hyman et al. (Mol. Biol. Cell 3, 1155-1167, 1992) with the rate of GTP hydrolysis on the plus end of MTs stabilized with slight modifications. This weakly hydrolyzed analog readily induces taxol and a slowly hydrolizable GTP analog, GMPCPP. When porcine brain PC-tubulin to assemble into microtubules in 0.1 M these stabilized microtubules are incubated with 32P labeled GTP PIPES, pH 6.9, 1 mM Mg 2, 2 mM EGTA, and upon the addition of 2 approximately 10 molecules per end are incorporated into the M glycerol, induces cold-stable ribbons and microtubules. In the microtubules. These GTP molecules are hydrolyzed quite slowly presence of 2 M glycerol, assembly is nearly temperature independent (10-30 % hydrolysis in 20 minutes). We are currently using this between 10-370C (similar to taxol-induced assembly), and even at 40C assay to identify factors that may regulate the rate of GTP the cc = 0.3 mg/ml. In the absence of glycerol, van't Hoff analysis hydrolysis at the ends of MTs. Previous studies have suggests thermodynamic parameters similar to taxol-induced assembly demonstrated that mitotic MTs have a much faster catastrophe except with a heat capacity change similar to GTP-induced assembly. rate than interphase MTs in Xenopus laevis egg extracts Investigations of the linkage between Mg*2 binding to GMPCPP and (Belmont et al, Cell 62:579-589). One way this may be regulated GMPCPP binding to tubulin are currently underway (Correia et al. J. is by increasing the rate of GTP hydrolysis in mitotic extracts. Biol. Chem. 262, 17278-17284, 1987). The enhanced stability of nuclei, Consistent with this, we have found that stabilized MTs in high even at 40C, suggests GTP is normally hydrolyzed during nucleation speed supernatants of mitotic extracts have a higher rate of leading to reduced nuclei stability. This result is consistent with a GTP hydrolysis than those incubated in interphase extracts. small GTP--cap size on a growing microtubule and favors the Lateral Separation of mitotic extracts on a sizing column reveals two Cap model (a single GTP-tubulin layer) for microtubule assembly peaks of activity that increase the rate microtubule GTP (Bayley etal., J. Cell Sci. 95, 33-48, 1990). Enhanced nuclei stability hydrolysis. One co-fractionates with a solution GTPase activity, further suggests the ability to study nuclei size, structure, and assembly the other does not. The activity binds weekly to pathways with this weakly hydrolyzable GTP analog. Initial studies phosphocellulose and can be separated from activities that bind by EM and analytical ultracentrifugation (on a Bseckman Optima tightly to MTs. XL-A) are being pursued. [Supported by GM41117 (J.J.C.).]

955 956 ATP-INDUCED BREAKDOWN OF STABLE MICROTUBULES IN EXTRACTED THE ENDS OF STABLE. DETYROSINATED MICROTUBULES ARE CELL MODELS. ((A. S. Infante and G. G. Gundersen)). Department of Anatomy & Cell REFRACTORY TO SUBUNIT ADDMON IN EXTRACTED CELL MODELS. Biology, Columbia University, New York, NY. ((M. Stein and G.G. Gundersen)) Department of Anatomy and Cell Biology, Columbia University, New York, NY. The generation ofstable, detyrosinated microtubules (Glu MTs) has been correlated with a number ofmorphogenetic events, yet the factors controlling the formation and breakdown In fibroblasts and epithelial cells. stable microtubules enriched in detyrosinated tubulin ofthese MTs are not known. To address the mechanism ofstable MT breakdown, we (Glu MTs) do not add or lose tubulin subunits at their distal ends (PNAS 84.9040 examined the sensitivity ofGlu MTs in extracted TC-7 monkey kidney cells to a variety of [19871). suggesting that their stability results from end-capping. We have developed an reagents. We used a standard immunofluorescence protocol with antibodies to tyrosinated extracted cell model to study in detail the growth properties of the ends of stable Glu (Tyr) and Glu tubulin to determine the effects ofthe treatments. After extraction ofTC-7 MTs as well as dynamic tyrosinated (Tyr) MTs. African green monkey kidney cells cells with 0.2% TX-100 and incubation in PEM (PIPES, EGTA, MgCI,), most Tyr (TC-7) were extracted briefly in 200 sg/ml saponin in PEM (0.1 M PIPES. 6.9, 1 mM (dynamic) MTs depolymen7e within 1S min. while most Glu MTs remain intact for over I EGTA and I mM MgCI,) and then incubated for 2 min in PEM containing 0.12 mg/mi hr. When cytoskeletons were incubated in PEM with 100 pM ATP -90YO ofthe Glu MTs DEAE-purified brain tubulin and I mM GTP before fixation and immunofluorescence. were lost within 1 hr. Incubations with phosphate. pyrophosphate. AMP, ADP, or the The exogenous tubulin was detected with an antibody reactive with a brain-specific nonhydrolyzable analogues AMP-PCP or AMP-PNP did not stimulate Glu MT breakdown. isoform of J-tubulin (antibody was a gift of R. Luduefia) and compared with the ATPh S was as effective as ATP, whereas GTP or UTP required a 10-fold higher distribution of Tyr and Glu MTs revealed by specific antibodies. We found that when concentration for an equivalent result. Both taxol and glycerol blocked the breakdown of brain tubulin was added immediately after extraction, -20-25% of the Tyr MTs Glu MTs induced by ATP. suggesting that ATP may act to remove a cap on Glu MTs (see incorporated brain tubulin at their ends (i.e., they grew) whereas 0%o of the Glu MTs abstract of Stein and Gundersen) thus allowing normal subunit exchange and MT grew. Higher concentrations ofbrain tubulin or the addition of recombinant tau slightly disassembly. Consistent with this. we found that Glu MTs exposed to ATE in the presence increased the percentage of growing Tyr MTs, but not Glu MTs. In attempts to induce ofglycerol disassembled when glycerol and ATP were removed. Our results support the subunit addition from the ends of Glu MTs. we treated cytoskeletons that had been idea that a protein kinase is responsible for triggering the breakdown of Glu MTs. In stabilized with taxol or glycerol. with a number of agents known to breakdown stable order to identify this kinase, we attempted to block the ATP effect with specific kinase Glu MTs (e.g., ATP. Ca", high salt; see abstract of Infante & Gundersen); none of inhibitors (e.g.. staurosporine, K252a. genistein, heparin, DMAP), but to date we have not these treatments enabled Glu MTs to grow. Several of these treatments made the Glu been able to inhibit the ATP effect. We did find, however, that two tyrosine phosphatase MTs susceptible to fragmentation, and in the case of Ca'nmany of the resultant inhibitors (vanadate and PAO) partially inhibited the ATE effect. This raises the fragments incorporated exogenous tubulin on one end. Our results provide further possibility that tyrosine dephosphorylation. perhaps ofthe putative kinase, is necessafy for evidence that Glu MTs are capped and suggest that soluble cytoplasmic factors may be stimulating Glu MT breakdown. In summary, our resuts show that stable Glu MTs can be necessary to uncap them. Supported by NIH GM42026. destabilized by an ATP-induced process that is likely to involve a protein kinase which is closely associated with the MTs.

957 958 A TIME SERIES APPROACH TO CHARACTERIZNG MICROTUBULE DYNAMICS. MICROTUBULE DYNAMIC INSTABILITY IS A THREE-STATE PROCESS ((DJ. Odde and H.M. Buettner)) Deptment ofChemical and Biochemical Engineering, ((P.T. Tran & E.D. Salmon)) Biology Department, University of North Carolina, Rutgers University, Piscataway, NJ 08855. Chapel Hill, NC 27599-3280. (Sponsor by A.K. Harris) Microtubules are dynamic protein filaments which undergo extendedperiods of The GTP-tubulin cap model predicts that cuttinig microtubules in the middle will produce approximately linear growth and shrinkage with abrupt transitions between these two states. exposed plus and minus ends that would rapidly shorten because the labile core of GDP- This dynamic behavior is most often chractemied by the mean growth and sringe rates tubulins are no longer stabilized by a GTP-tubulin cap. However, when microtubules and the mean growth and shrinkage times. However, defining growth and shriniage phases assembled from pure mammalian brain tubulin were cut with a UV microbeam, plus severed is subjctive, making the characterization ofdynamics difficult when the behavior is ends rapidly shortened, but minus severed ends did not shorten but resumed growth (Walker nonideal. More generaly, experimentally obtained microtubule length life histories can be et al., 1989, J.Cell.Biol., 108(3):931-937); a result not predicted by the GTP-tubulin cap regardod as complex signals in time. Such complex signals are routinely characterized in model. We tested the possibility that UV irradiation somehow modifies severed minus ends engineering using time seris analysis. Therefore we simulated microtubules using the by mechanically cutting individual microtubules. The polymerization dynamics of idealized behavio ofmicrotubule dynamic instability adcbhactrzed the dynamics by time individual microtubules grown from axonemal seeds were recorded by high resolution video series analysis. Specifically, length life histories were simulated assuming constant growth microscopy techniques while they were cut by pressing them against the coverslip surface and shrinkage rates coupled with random selection of growth and shrinkage times from with the tip of a glass needle whose position was controlled by an Ellis piezo-electric gamma probability distributions. A basic parameter set was chosen, 10 gam/min growth and micromanipulator. At 16 uM tubulin, the percentage of ends which rapidly shortened was shrindge rates and 60 sec mean growth and shrinkage times, and then varied over several 92% (n=227) for severed plus ends and 9% (n=123) for severed minus ends. Raising the free orders of magnitude. Net microubule displacements wer caculatedover discrete time tubulin concentration to 32 uM reduced the percentage of ends which rapidly shortened to intervals and charatrzed using the power spectum as well as the sutcr and 42% (n=64) for the severed plus ends and 1% (n=76) for the severed minus ends. We partial autocorelation functons. Simulated microtbule showed significant autocosrelation conclude that the two-state GTP-tubulin cap theory is insufficient to explain microtubule and periodicity dupending on the sampling rate and dynamic parametes. The time series dynamic instability, particularly for minus ends. From our results we propose a three-state analysis was sensitive to variations in the growth and shrinkage probability distributions model of dynamic instability: and the mean time spent in one phase relative to the other but not to the growth and kl k3 shrinkage rates. Since there are many microtubules in a cell we simulited microtubule G <===> I <=> S populations and found the time series behavior was not significantly different from that of a k2 k4 single microtubule with the same parameter set. The time series analysis results obtained where the growth (G) state is stabilized by GTP-tubulin addition, the intermediate (I) state here for idealized microtubues can now be radily compared to the time series analysis of represents the state of severed ends immediately after cutting, and the shortening (S) state experimental length life histories. Thus, time series analysis provides an objective and corresponds so rapid GDP-tubulin dissociation. For plus ends, k3>>k2 while for minus quantsttive basis for mirotubule dynamic chactrzation and applying it to both ends, k2>k3 at 16 uM tubulin, anid k2 is dependent on tubulin concentration for both ends. experimental and simulated dataenhances our ability to discriminate between competing The differences in the rate constants between plus and minus ends may be related to the models of microtabule dynamic instability. orientation of the dimeric lubulin subunits as the ends of the microtubule lattice. (Supported by NIH GM 24364) Monday. Microtubule Dynamics and Assembly II (959-961) 165a

959 960 MICROTUBULE ASSEMBLY INHIBITION BY PORPHYRINS. ((I.Ringel', L. MICROTUBULE DYNAMIC INSTABILITY OF SUBTILISIN CLEAVED Levdansky', V. Gottfried2, and S. Kimel2)) 'Department of Pharmacology, TUBULIN ((L Cassimeris)) Dept. of Molecular Biology, Lehigh University, Faculty of Medicine, The Hebrew University, Jerusalem and 2Department Bethlehem, PA 18015 of Chemistry, Technion, Haifa, Israel. The highly charged carboxy termini of a and B tubulin have been implicated as Porphyrins and porphines analogs have been shown to induce cytotoxic important functional domains for microtubule (MT) dynamic instability (FEBS. 271: 1-8). To test this idea, the carboxy termini of phosphocellulose-purified effects on cells and tissues. This effect is enhanced dramatically with porcine brain tubulin were removed by cleavage with the enzyme subtilisin. exposure to light (PDT). One of the major targets proposed for the action Two preparations were generated: native a, cleaved B (ais) and cleaved a and B of porphyrins is tubulin. In our study we analyzed the potency of TPPS4, (asBs). Blotting with antibodies against the carboxy termini showed that TPPS2, o-,m-,p-THPP, as inhibitors of tubulin assembly. Without TPPS,, subtilisin cleavage removed < 15 amino acids from a and B tubulin. About any exposure to light, was found to be the most potent inhibitor, TPPS, 20% of B tubulin was also cleaved internally to yield fragments of -32 and 18 followed to a lesser extent The THPP by TPPS, and much by TPPS4. kD. aB8s assembled into MWs that elongated from both ends of axoneme analogs have negligible inhibitory effect Upon exposure to light, all fragments. The dynamic instability of aBs (21 pM tubulin at 23°C) was as analogs induced inhibition of tubulin assembly follows: indistinguishable from uncleaved tubulin with one major exception: aBs TPPS2,>TPPSh.>TPPS4>THPP. All analogs were found to possess high shortened 2.7 times faster at the plus end compared to uncleaved tubulin (mean affinity to the same site on tubulin, even those that failed to inhibit velocities: 48 and 18,um/min, respectively). asBs assembled into MT and assembly without light (THPP). Binding to monomeric tubulin is nonMT structures (Arch. Biochem. Biophys. 279: 328-337.) and elongated mandatory for effective inhibition of assembly with or without exposure to from both ends of axoneme fragments. Because asBs assembled into rings, light. Addition of porphyrins to assembled microtubules does not cause open sheets, etc., its assembly kinetics were not analyzed in detail. But, their depolymerization even with prolonged exposure to light. It was dynamic instability behavior was observed and the mean shortening velocity proposed that porphyrin analogs may share the same binding site on appeared faster at both ends (mean = 59 pm/min). The results with aBs suggest tubulin as bis-ANS, a known tubulin assembly inhibitor. Our competition that altering the intra-dimer bond (B C-terminus to a N-terminus; EMBO 4: studies between the porphyrin analogs and bis-ANS, suggested that the 2397-2402) alters the off rate constant during shortening specifically at the plus analogs do not share the same site as bis-ANS. end. The results with aBss suggest: 1. Altering both the intra- and inter-dimer protofilament (B N-terminus to a C-terminus) bonds causes significant structural changes in the MT lattice, but does not eliminate dynamic instability; 2. the carboxy termini of a and B tubulins are not solely required for the structural change in the MT lattice believed necessary for dynamnic instability.

961 THE PARADOXICAL EFFECTS OF LOW CONCENTRATIONS OF NOCODAZOLE ON MICROTUBULE DYNAMIC INSTABILITY: STABILIZATION INDUCTION AND CATASTROPHE PROMOTION ((R.J. Vasquez and L. Cassimeris)) Dept. of Molecular Biology, Lehigh University, Bethlehem, PA 18015 Previous studies by Jordan et al. suggest that the anti-mitotic effects of the drug nocodazole may be attributed to stabilization of spindle microtubules (MTs) rather than depolymerization of the MTs (J. Cell Sci. 1992. 102: 401- 416). Following on this work, we are using video-enhanced DIC microscopy to characterize the effects of low concentrations of nocodazole (4nM - 400nM) on the dynamic behavior of phosphocellulose-purified porcine brain tubulin assembled from axoneme fragments isolated from sea urchin sperm. Preliminary data suggest these concentrations of nocodazole have contradictory effects on the dynamic behavior of individual MTs. At the concentrations tested, nocodazole appears to stabilize MTs at both plus and minus ends by causing varying periods of no detectable elongation or shortening (time periods range from 1-2 sec during shortening up to 300 sec during elongation) and by decreasing the shortening rate -50% compared to control data (eg. for minus ends, 7-17 vs. 29jum/min.). These preliminary findings are in agreement with published data from similar experiments on another antimitotic drug, vinblastine (Toso et al. 1993. Biochem. 32: 1285-1293). However, in addition to this increased stability, low concenMtrations of nocodazole also cause a 3-fold increase in plus end transitions from elongation to shortening (catastrophe) compared to controls (1 catastrophe every 37 sec vs 1 every 112 sec ). The data suggest a potential dual role for nocodazole as both a stability inducer and a catastrophe promoter. The plus end catastrophe promoting activity may mimic a cytosolic component which generates the higher catastrophe frequency observed in vivo. Cell Motility II (962-963)

962 963 MORPHOLOGICAL TRANSFORMATION OF HUMAN PLATELETS QUANTIFICATION OF CELL SHAPE CHANGES IN THE AS VIEWED BY VIDEO-ENHANCED DIC MICROSCOPY. EARLY DEVELOPMENT OF THE CHICK THYROID. ((G.M. ((D.J. Loftus and S.J Smith)) Department of Molecular and Cellular Kinebrew and S.R. Hilfer)) Department of Biology, Temple Physiology, Stanford University School of Medicine, Stanford, CA University, Philadelphia, PA [9122 94305. The early chicken thyroid gland develops as a series of concentric ovoid rings that evagmates from the ventral floor of the pharynx. The normal functioning of platelets involves transformation from a Previous work has shown that growth of the thyroid primordium "resting" state to an "activated" state upon exposure to a variety of occurs predominantly as a result of recruitment of cells from the physiological stimuli. We have used the technique of video-enhanced pharynx. Time-lapse video recordings of cell movements indicate that cells in the region of nearest the behaviors differential interference contrast (DIC) microscopy to observe, in real the thyroid pharynx display consistent with cell sorting and cellular rearrangement. Work done on time, the morphological transformation that occurs in single platelets that other systems has consistently shown that cells accom.plish have been activated. At rest, platelets are discoid and have smooth rearrangement by changing shape. In order to ascertam whether or surfaces, lacking cytoplasmic projections. Upon exposure to thrombin or not this is an active process involving constriction of circumapical other potent activating agents, platelets undergo spreading and edge microfilament bund[es in this system, we measured the circumference time. The living at ruffling. Activated platelets also develop numerous filopodia, which of the cells over thyroid primordium developmental stage 14 was exposed and maintained in Hank's saline. extend and retract. In more movements are rapidly addition, complex Fluorescently labeled phalloidin was incorporated into the cell apices seen, such as filopodial "waving", bending and folding of filopodia and by iontophoresis. Time-lapse recordings of the thyroid preparations filopodial bifurcation. Although platelet activation has been studied tracked the cells for as long as the fluorescent signal could be extensively by electron microscopy and other methods, the studies maintained. It was found that cells near the thyroid/pharyngeal border here reveal additional information about the of these do change their apical diameter and that this appa to be due to reported dynamics constriction of circumapical microfilament bundles. Ongoing processes that is not evident by other techniques. investigations are focused on further documentation that the types of cell shape changes associated with cellular rearrangement do occur in this system and whether or not cells in more central regions of the thyroid utilize constriction of circumapical filaments to facilitate shape changes associated with cellular rearrangement. 166a Cell Motility 11 (964-969). Monday

964 965 DISRUPTION OF IN GROWTH CONES OF LEECH THE INDUCTION OF PROTRUSION BY PDGF AND NEOMYCIN IS CORRE- INDUCED BY DEPOLARIZATION AND CALCIUM INFLUX. LATED WITH A DECREASE IN FILAMENTOUS AC`lN. ((B. Safleko-Mroczka, K. ((M.D. Neely and M. Gesemann)) Biocenter Univ. Basel, 4056 Basel, Hedber& B.-A. Fredriksson, M. Lindroth, and P.B. Bell, Jr.)) Department of Zoology, Switzerland The University of Oklahoma, Norman, OK 73019, USA; and Department of Pathol- ogy Linkoping University, S-581 85 Link6ping, Sweden Depolarization of leech neurons leads to loss of filopodia, rounding-up of peripheral regions of the growth cones and finally to neurite retraction. This move a response is influenced by the substrate on which the cells are growing and is Metazoan celis by three-phase procesa that involves protusion of the cell mediated by the depolarization-associated influx of calcium (S. Grumbacher- periphery, establishing adhesions between the protruion and the substratum, and Reinert and J. Nicholls, J. Exp. BioL 167:1-14, 1992; M.D. Neely, J. Neurosci. withdrawing the protrusion. Although thought to involve changes in the organiza- 13:1292-1301, 1993). We have analyzed possible changes in cytoskeletal tion of actin, the mechaniama responsible for protusion are stil unclear. We have in- organization that accompany these morphological changes. Immunocyto- vestigated the mehansm of protnion using platelet derived growth factor (PDGF) chemical analysis of leech neurons growing on leech extracellular matrix and neomycin, an inhibitor of the phosphatidyHinositol cycle, to stimulate protrusion extract revealed a loss of the growth cone microfilaments after depolarization. in human fibroblasts and glioma cells. Changes in cell motility and the cytoakeeton Stabilization of microfilaments with phalloidin inhibited depolarization-induced were examined by video, fluorescence, and scanning electron microscopy and neurite retraction, whereas disruption of microfilaments with cytochalasin cytofluorometry. PDGF stimulates both protrusion and withdrawal of the cell pe- caused a response similar to the retraction observed after depolarization. Leech riphery, whereas neomycin stimulates only protrusion. In combination PDGF and neurons growing on a different substrate, the plant lectin concanavalin A, do not neomycin stimulate first protrusion, followed by a to a PDGF-like pattern of retract their neurites after depolarization, but continue to grow (S. Grumbacher- change protrusion and withdrawal. Protrusion in both PDGFand neomycin is preceded by a Reinert and J. Nicholls, 1992; M.D. Neely, 1993). This is likely due to the much reduced calcium influx during depolarization of neurons growing on this rapid decrease in the amount of F-actin in the cell. These results are consistent with substrate (Ross et al, PNAS 85:4075-4078, 1988). Increasing the intracellular the hypothesis that protrusion is caused, at least in part, by severing and depolymer- calcium concentration by incubating these neurons with the calcium-ionophore ization of F-actin. Withdrawal is correlated with an increase in F-actin, consistent A23187 led to cessation of motility and disruption of microfilaments in growth with the hypothesis that withdrawal requires polymrization of actin filaments in the cones. As judged by immunofluorescence, microtubule organization was not newly protruded lamella. PDGF- and neomycin-induced changes in the organization affected by this treatment. We have observed colocalization of microfilaments and amount of F-actin are paralleled by changes in the distribution and amount of and an antigen that cross-reacts with antibodies against , a calcium- gelsolin. A nmdel is proposed that links the PDGF- and neomycin-induced release of activated microfilament-severing protein and are presently analyzing the nature gelsolin from its association with phosphatidylinositol 4,5-Aphosphate (PIP2) at the of this antigen with the goal of establishing its potential role in the calcium- cytoplasmic face of the plasma menbrane with the severing of F-actn and induced disruption of microfilaments in leech neuronal growth cones. the initia- Supported by the Swiss Nationalfond No. 31 27 814.89 tion of protrusion. (Supported by the University of Oklahoma and the Swedish Cancer Society - grants #2212 and #2985).

966 967 CELL MOTILITY DURING WOUND HEALING IS TRIGGERED BY THREE-DIMENSIONAL MEASUREMENTS OF CALCIUM DISTRI- INTRACELLULAR CA2+, AND IS STIMULATED BY BRIEF LI+, BUT BUTIONS IN MOTILE FISH EPIDERMAL CELLS USING TWO- NOT SERUM ADDITION. ((P.J. Sammak, G.E. Moeller, B.I. Trinh, and PHOTON EXCITATION FLUORESCENCE MICROSCOPY. R.R. Speck)) Dept. Pharmacology, U. Minnesota, Minneapolis, MN 55455. (I. Brust-Maschert, R.M. Williams*, and W.W. Webbt)) tApplied & Eng. Physics and *Physics, Cornell University, Ithaca, NY 14853. Cell locomotion in confluent monolayers is induced by wounding, but the Internal calcium concentrations were measured in fish keratocytes, whichi intracellular signals that initiate or maintain motile behavior are not known. A extend broad, submicron thin, actin-rich lamellipodia and migrate rapidly wave of intracellular free Ca2+ (Ca2+] j) was observed in bovine pulmonary in culture with their cell bodies located at the rear. 3-dimensionlal sub- endothelial cell wounds. The [Ca +]Ji wave stimulates movement in the absence micron spatial resolution was obtained by two-photon ultraviolet excita- of exogenous growth factors. A 2 minute addition of Ca2+ channel blockers tion using a red, mode-locked, 100 femtosecond pulsed laser (Denk et al., (Co2+, Cd2+, Gd3+, or SKF96365) during wounding reduced long term 1990). Cells were loaded with the calcium indicator Indo-I AM, and in- motility by half. Agents that interfere with release ofCa2+ from intracellular tracellular calcium activity was determined using the fluorescence ratios stores (U73122, an inhibitor of phospholipase C, and thapsigargin, an inhibitor to eliminate dye concentration and cell thickness errors. Migrating cells of the Ca2+ -ATPase) also reduced motility. Brief addition of reversible in- were found to exhibit concentration heterogeneities with calcium levels 1 hibitors of Ca2+ stores release (cyclopiazonic acid) and influx (Co2+), blocked to 3 times higher in the perinuclear volume at the rear of the cell. Con- motility further. Therefore, both intemal Ca2+ stores and extracellular Ca2+ are centrations in the lamella were typically 200-400 nM. The size of these employed at the start of wound-induced motility. Brief addition of 1 AM Li+ overall gradients showed no correlations to the speed with which the stimulated motility 2 fold for the duration of wound closure. Maximal stimula- cells were moving. However, non-uniform calcium distributions within tion by Li+ (3 fold), was more than the maximal stimulation by insulin or the lamella did show correlations to cell motion; turning cells tended serum (2 fold). If Li+ was applied 20 min before or after wounding, there was to pivot around regions in their lamellae which were higher in calcium. no stimulation of motility. Therefore, Li+ only stimulates during an initiation When subjected to an applied electric field, the same behavior was ob- step for motility and does not prime or maintain cell motility. In contrast, serum served as the cells turned toward the cathode. does not trigger motility at an initiation step. Instead, serum stimulated motility Supported by rants from NSF and NIH to the Developmental Resource in proportion to the duration of treatment. Neomycin, which inhibits inositol for Biophysical Imaging and Opto-Electronics. signaling, does not slow motility in the absence of Li+ and serum. Perhaps Li+ acts acutely by reducing inositol (1,4,5) trisphosphate turnover. The activity of exogenous Ca2+ and Li+, but not serum, during the initiation of movement might identify the [Ca2+]i inositol signaling pathway as a trigger for motility. The trigger determined speed throughout wound closure, and was distinct from serum stimulation which affected maintenance, not initiation of motility. (AHA)

968 969 VISUALIZATION AND MEASUREMENT OF CELL-SUBSTRATUM CELL-SUBSTRATUM ADHESION PATTERNS ARE RAPIDLY TRACTION FORCES GENERATED BY FISH KERATOCYTES. ((T.N. ALTERED BY ELECTRIC FIELDS. ((MJ. Brown and LM. Loew)) Oliver, J. Lee, A. Ishihara, M. Leonard and K. Jacobson)) Dept. of Cell Department of Physiology, University of Connecticut Health Center, Biology and Anatomy, University of North Carolina at Chapel Hill, NC 27599. Farmington, CT 06032. We have observed a distinctive pattern of traction forces generated byrapidly locomoting cells on a flexible substratum and we have now measured these NIH/3T3 fibroblasts exhibit cathode directed migration when exposed forces by two means, using calibrated microneedles. First, we modified the to DC electric fields. We have been testing the hypothesis that the silicone rubber traction assay developed by Haris et al. (1980), so that Iljm galvanotactic response of these cells involves electric field induced latex beads embedded in a weakly crosslinked silicone rubber film would reveal gradients of cellular adhesiveness. Using video enhanced interference deformation of the substratum in two dimensions. Keratocytes moving at -0.3 reflection microscopy (IRM), we have examined the effects pm/sec. exert traction forces on the silicone rubber sufficient to move thebeads, of applied but do not appear to wrinlde the film. The pattern of traction force vectors electric fields on cell-substratum adhesion patterns. Electric fields as displayed by a field of such bead displacements is symmetric about the cell's low as 0.1mV/jum induce a rapid, cathode-directed increase in cell- midline for a keratocyte moving along a straight path. We see the largest bead substratum proximity. At field strengths oflmV/sm adhesion patterns displacements (5-10 at the rear lateral margins of the cell's lamella, gm) are dramatically rearranged within 5 to 10 seconds, preceding any orientated perpendicular to the cell's axis of locomotion. Moreover, the tracdon apparent morphological change or cell movement. If the field is turned forces around the cell's perimeter appear graded in a manner consistent with the Graded Radial Retraction model of locomotion described for this cell by Lee et off immediately after this initial phase, the distribution of contacts al., 1993. Using microneedles to reproduce such bead movements in the elastic relaxes to the prefield pattern. Continuous field exposure leads to cell film, we estimate the force required by the cell to deform the substratum to be polarization and directional migration, accompanied by a cathodal -0.5 mdyne/tm. In a second approach, the maximum traction force exerted by increase of cell-substratum contacts and decreased anodal adhesion. the whole cell was estimated (-6 mdyne) by measuring the deflection of a Our results suggest that the galvanotactic migration of these cells may microneedle positioned in the path of keratocytes locomoting on a more rigid be mediated by electric substratum. Supported by NIH GM 31325. field-induced asymmetries of cell-substratum adhesion. (supported by USPHS grant no. ES05973) Monday. Cell Motility II (970-975) 167a

970 971 CELL CYCLE-DEPENDENCE OF HL-60 CELL DEFORMABILITY STUDIES ON THE GENESIS AND STABILITY OF NUCLEAR ((M. A. Tsai, R. E. Waugh, and P. C. Keng)) Department ofBiophysics, SEGMENTATION IN HUMAN NEUTROPHILS. ((M.S. Campbell, University ofRochester Medical Center, Rochester, NY 14642 M.A. Lovell, and GJ. Gorbsky)) Dept. of Anatomy and Cell Biology, University of Virginia Health Sciences Center, Charlottesville, VA Leukocyte mechanical properties significantly influence blood flow and 22908. oxygen delivery in the microvasculature, and play an important role in cell retention in the capillaries. In the present study, we invesfigated changes in The nucleus of the mature human neutrophil is segmented into 3-5 cellular mechanical properties during cell cycle, using human promeylocytic interconnected lobes. The physiological purpose of this segmentation is leukemic cell line (HL-60) as a model. HL-60 cells in G1 and S phases were unknown as is the mechanism by which the lobes are formed during separated by centrifugal elutriation and the cell cytoplasmic viscosity was differentiation. Using video observation of migrating human neutrophils evaluated by single-cell micropipette aspiration. The viscosity was calculated simultaneously illuminated for fluorescence and phase contrast micro- according to a numerical analysis ofthe cell entry into the micropipette (Tsai et scopy, we analyzed nuclear movements with respect to cell shape al, 1993). HL-60 cells in S phase were found to be less deformable than cells changes. The number of nuclear lobes and their relative size remained in GI phase. The cytoplasmic visosities of GI and S cells were found to be constant during observation (up to 1 hr). The thin segments inter- from 450 Pas to 160 Pas and from 50 Pas to 25 Pas respectively, when connecting the lobes elongated and attenuated extensively but never tested at aspiration presures ranging fiom 294 Pa to 882 Pa. Both groups of separated. HL60 cells are a human promyelocytic line that, with retinoic cells exhibited non-Newtonian behavior, in that the apparent viscosity acid treatment, segment their nuclei and differentiate into neutrophil-like decreased with increasing shear rate, similar to normal neutrophils. The cells over several days. Using a rapidly responding variant line termed dependence of the cytoplasmic viscosity on defonnation rate can be described HL60/S4 (Leung et al., Cancer Res. 52:949-954), we found that segmentation could be induced within 24 hr. We tested the role of cyto- empirically by = Lc( IycYb, where p is cytoplasmic viscosity, im is skeletal elements in the process of nuclear segmentation. Neither the mean shear rate, ji, is the charcteristic viscosity at the characteristic shear rate microtubule inhibitor nocodazole nor the microfilament inhibitor cyto- yC, and b is a material coefficient When ic was setto 1 s-',.y=76 Pas and b=0.42 for G1 cells, and &=300 Pas and b=0.47 for S cells. These results chalasin D prevented nuclear segmentation. Together our studies sug- gest that nuclear lobes in neutrophils are relatively stable structures that provide detailed quantification of the dependence of leukocyte mechanical are not generated by niicrotubule- or microfilament-dependent forces. properties on cell cycle.

972 973 REGULATION OF (MSP) CYTOSKELETAL MSP FROM THE AMOEBOID SPERM OF ASCARIS FORMS A DYNAMICS IN THE AMOEBOID SPERM OF ASCARIS. ((T.M. Roberts, HIERARCHY OF HEUCAL COMPLEXES RELATED TO AMOEBOID K.L. King, J. Essig, and T.S. Moorland.)) Department of Biological Science, MOTILITY. ((K.L. King', M. Stewart2, and T.M. Roberts.)) 'Florida State Univ., Flonda State University, Tallahassee. Dept. of Biol. Sd., Tallahassee, FL., 2MRC, Lab. of Mol. Blol., Cambridge, UK.

The amoeboid migration of Ascaris sperm depends on vectorial assembly of AMaris sperm are unusual amoeboid cells that lack actin and . Instead, their cytoskeleton Is composed of major sperm protein (MSP). Self-assodation of a unique MSP cytoskeleton whereby filaments form preferentially along the monomers into helical is an property of leading edge of the pseudopod and disassemble at the cell body-pseudopod assembles Intrinsic MSP. This feature is clearly displayed in the number of polymorphic forns MSP assumes in vivo and In junction. This pattem of cytoskeletal rearrangement requires precise control vitro. We have characterized 4 disfnct assembles-subfilaments, filaments, of MSP polymerization which appears to be mediated, in part, by intracellular macrofibers, and fiber complexes-each having one or more helcal components. pH. Fluorescence ratio imaging of sperm loaded with the pH-sensitive dye, Indhivdual filaments assembled either In vivo or In vitro are composed of two BCECF, revealed a pH gradient from 6.4 at the leading edge, where filaments subfilaments wound together as a right-handed helx 10 nm wide. Some filaments assembled, to 6.2 at the base of the pseudopod, where disassembly occurred. have single or separated subfilaments at their ends. Filaments assembled In vitro Acidification of these cells caused complete disassembly of the cytoskeleton. coil around one another to form larger helical assembles, macrofibers, that do not Removal of the acid allowed pHi to rebound and resulted in reassembly of the require accessory proteins to bundla. MSP filaments in vivo also coil around each MSP filament system along the pseudopod membrane. This sensitivity of other to form fiber complexes. Overal, the macromolecular assembles formed by filament formation to pH, was also observed in earlier stages of MSP are analogous to a rope: Individual polypeptide chains polymerize to form . , which contain MSP In paracrystalline subfilaments; two subfilaments coil around each other to form filaments; and fila- fibrous bodies, exhibited pH1=6.8. After , decreased to 6.2 and ments coil around each other to form macrofibers and fiber complexes. pH, The MSP system In sperm operates much Ike the actin cytoskeleton In other the fibrous bodies disassembled so that the haploid had no spermatids amoebold cells: filaments form along the leading edge and detectable MSP filaments. Activation of spermatids to mature into motile organize into 3-D arrays that flow rearward as the cell crawls. Self-associatIon of MSP filaments into a spermatozoa triggered rapid increase in pH, to6.4 and concomitant helcal superstructures may have important Impicatfons for the role of MSP In reassembly of MSP. Treatment of these cells with weak bases induced the sperm motflty. This property could allow filaments to form fiber complexes without same rapid formation of filaments. Filament assembly at all stages of requiring the acoessory proteins needed to Ink F-actin and, thus, create a simpler development was associated with elevated pH, and occurred preferentially framework to propel locomotion. at membranes. Thus, sperm appear to control filament formation by pH- sensitive MSP-membrane interaction. Supported by NIH GM29994.

974 975 GENETIC ANALYSIS OF THE ROLE OF A MAPKINASE (ERK) IN CYCLIC AMP POLARIZES HEL CELLS AND INDUCES MOTILITY. CHEMOTAXIS AND AGGREGATION OF DICYOSTELIUM ((M. Y. Niu*, J. C. Mills+ and V. T. Nachmias*)), *Department of Cell and DISCOIDEUM. Developmental Biology and Department of Pharmacology+, ((.E. Segall, A. Kuspa*, C. Gaskins, G. Shaulksy*, M.E. Ecke, M. University of Maeda*, R. Firtel*, and W.F. Loomis*))Department of Anatomy and Pennsylvania, Philadelphia, PA, 19104. Structural Biology, Albert Einstein College of Medicine, Bronx, NY Human erythroleukemia (HEL) cells grow 10461, *Center for Molecular Genetics and of rapidly in suspension. They spread Department on when treated Biology,University of California, San Diego, La Jolla CA 92093 fibronectin with nM phorbol myristate acetate (PMA) as reported by Jarvinen et al., Eur. J. Cell Biol. 44,238,1987). We found that 10 nM PMA caused Factin to increase to 127+/-10% of control if cells are kept Dictyosteliwn amoebae are chemotactic to folate during growth suspended, increasing to 186+/-21% if cells were allowed to spread. and use chemotaxis to cAMP to aggregate cells. Restriction Enzyme Stress fibers ending in and patches appear in the spherical, spread cells. Mediated (REMI) was used to generate a mutant defective in Intgration When PMA is washed out and dibutyryl cAMP is added, the cells aggregation and altered in chemotactic responses. The disrupted gene slowly polarize. Small cells with a typical ruffle appear by day 1 (dagC) was found to be similar tomap kinases and and larger, elongated (ERK1 ERK2). cells with smooth borders accumulate and Recapitulation of the insertion by homologous recombination in test dominate by day 2. Time lapse videomicroscopy shows that the smaller cells are actively motile, strains leads to a phenotype similar to that of the original mutant, while the larger elongated cells are indicating that the insertion is responsible for the loss of aggregation almost nonmotile. Stress fibres become faint in capability. Detailed studies indicate that the defect in chemotaxis is cell elongated cells while a prominent microtubule array radiates from the centrosome withspecific autonomous in mixtures of mutant and wild-type cells, mutant cells show visualized antibody (gift of E. Holtzbauer) and located reduced chemotaxis and altered localization during morphogenesis. The towards the longest arm of the cells. When nocodazole is added with the cAMP, themicrotubule array disappears, and elongation is inhibited. Time chemotaxis defect may be due to a reduced ability torespond to high lapse video shows unusualseaming in long tentacles andretacting from the concentrations of stimulus. These results indicate a role for map kinases in emergg cell body. We hypothesize that in association the regulation of cell movement and development. with microtubule elongation the motile smaller cells slowly alter into the elongated cells and lose their motility. Supported by: AR-40840. 168a Cell Motility II (976-978). Monday

976 977 LIGHT DEPENDENT EFFECTS ON DIATOM MOTLITY ((S.A. Cohn)) GLIDING MOTILITY & CELL SURFACE TRANSLOCATION EVENTS IN Department of Biological Sciences, DePaul University, Chicago IL 60614. SPOROZOAN PROTOZOANS.(C.A.King) Dept. of Biology,University College London WCIE.6BT,England. Diatoms are a ubiquitous type of aquatic golden algae, whose ability to move upward through the sediment during the day, and resettle into the sediment at Considerable interest has been shown in particle movement on the night, is thought to be an important factor in their ecological success. Such surface of crawling cells-however these studies are considerably light directed movements in other algae are often the result of a combination of complicated by the large scale reconstruction of the cytoskeleton light-dependent changes in speed of movement (photokinesis), orientation of during cell movement. By contrast the gliding motility of movement (phototropism), or reversal of direction at light/dark boundaries sporozoan zoites occurs without changes in cell shape but at fast (photophobic response). In order to detennine the relative contribution of each rates ( 1-10pm per sec.). Translocation of microbeads can be of these responses to phototaxis in diatoms, our lab is using computer-assisted demonstrated in several genera-Gregarina,Eimeria,and Plasmodium. video to microscopy quantify the motile activity of freshwater diatoms under Eimeria sporozoites have been studied in detailOvert gliding is different light conditions. Inital results suggest that diatom phototaxis is always in a forward direction and bead tr anslocation (plus substantially due to the photophobic response, as little effect of photokinesis is capping of surface receptors by ligands) is always from anterior observed. The average speed of dark adapted diatoms appears to be unchanged to posterior. It is proposed that a common cell surface linear at any illumination over the range of 10 lux to 10,000 lux, while the motor drives the motile processes. Inhibition of motility photophobic effect (as measured by the percentage of cells which reverse by cytochalasins, and the demonstration of actomyosin by direction when reaching a dark boundary) increases substantially with superprecipitation in cell extracts suggest actin-myosin illumination. At 10-15 lux only 15-20% of the cells change direction within one interactions provide the motive force. The classical experiment cell length of the boundary, while at 5,000 to 10,000 lux this percentage of myosin-coated beads moving on actin cables provides a model increases to 75-85%. This effect does not appear to be due to an overall for this system. It might be expected that the speed of bead increase in the frequency of changing direction in high illumination, since cells translocation on the Eimeria surface might be considerably slower constantly illuminated with 550nm light (no dark boundary) show no difference than that generally observed in the 'in vitro' motility assays in direction change frequency in 10 lux versus 7000 lux light (- 1 direction 1-3pjm per sec) due to the presence of the plasmalemma. However change / 75 ± 43 sec), while the photophobic response concurrently increases bead translocation rates in excess of 301m per second have been from 15% to 80%. This work suggests that diatom phototais is not generated recorded over short time intervals.Since sporozoan protozoans are by differenially tuming the motility apparatus on and off (since there is active obligate intracellular parasites, this motility system could movement at low light levels), but rather by biasing the direction of the provide the driving force to execute host cell invasion. movement into areas of bright illumination. This work was supported in part by a DePaul University Research Council Grant

978

STRUCTURE OF BACTERIAL ADHESION PILI - IMPLICATIONS FOR BACTERIAL PATHOGENESIS. ((E. Bullittl and L. Makowskil 2)) 'Boston Univ Physics and 'Florida State Univ, Inst ofMol Biophys, Tallahassee, FL 32306.

The 3D structure of P pili provides a basis for understanding the molecular strategies ofbacterial adhesion to epithelial cells. The structure of P pili from uropathogenic E. coli has been computed from electron microscopic data, providing information about the major structural protein, PapA. If PapA is a globular protein, there are two plausible assignments for the PapA monomer, with the PapA monomer comprising two globular structures. If, however, PapA is a filamentous protein, adjacent monomers have extensive regions of overlap, and each PapA monomer is highly elongated. Additional structural conformations of P pili are seen when the pili have been mechanically sheared. P pili are fairly straight helical structures, 68A in diameter and lpm in length. Under stress, pili can assume an extended form, called fibrillae, with a diameter of only -25A P pili are also seen with sharp bends, with the pili apparently unbroken at the turn. These conformations may aid E. coli in remaining bound to the host cell. In particular, we suggest that the fibrillae are a plastic deformation ofP pili. Plastic deformation thus provides a means by which bacteria can remain bound to epithelial cells under high shear conditions. Cytoskeleton-Membrane Interactions II (979-980)

979 980 CHARACTERIZATION OF PONTICULIN NULL CELLS: EVIDENCE THE MECHANISM OF INTERACTION BETWEEN ACTIN AND THAT PONTICULIN IS THE MAJOR ACTIN-PLASMA MEMBRANE MEMBRANE LIPIDS ((C. Gicquaud*; P. Wong**; J.F. Comtois*; LINK IN DJCTYOSTELlUM. ((A. L. Hitt and E. J. Worcester R. Grimard* and P. Trancrede*) *Departement de chimie-biologie, Luna)) Universite du Quebec, Trois-Rivieres G9A 5H7, Canada; **NRC, 100 Foundation for Experimental Biology, Shrewsbury, MA. 01545 Sussex Drive, Ottawa, Ontario KIA OR6, Canada)

Ponticulin is a well characterized integral that binds F-actin The current view stresses that the cytoskeletal protein actin is anchored to the membrane via intrinsic proteins. our and nucleates actin assembly in vitro. We have previously cloned the gene for However, laboratory has presented evidence that an interaction exists between actin and liposomes ponticulin from Dictyosteliwn discoidewn and have generated pontiulin null in conditions compatible with those existing in vivo. The present work is mutants by homologous recombination. Two independent clones are aimed at investigating the mechanism of interaction between actin and currently being analyzed. Preliminary analysis of the ponticulin null mutant membrane lipids. Differential scanning calorimetry reveals that the shows that it clears bacterial lawns and organization of the lipid molecules in the bilayer remains unchanged but undergoes apparently normal that the actin conformation is drastically altered during interaction. This development. Crude membranes from ponticulin-null cells have greatly change in actin conformation, as confirmed by FITR spectroscopy, results reduced amounts of associated actin and myosin by SDS-PAGE, as compared in more B sheets, in a stronger monomer-monomer interaction and in less to control plasma membranes. Highly purified membranes from the stability of the protein to high pressures. In addition, a monolayer study transformants do not nucleate actin assembly in vitro, nor do they bind actin indicates that at low surface pressures, actin intercalates into the bilayer up to a critical surface pressure above which actin interacts only with the polar in co-sedimentation assays. Cells lacking ponticulin appear to have less total heads. Interaction of actin with lipids requires divalent cations, is inhibited actin than parental cells, suggesting that actin expression has been down- by monovalent cations and is influenced by the polar heads of the lipid. A regulated. Further analyses of these mutants are underway, including model is presented in which divalent cations neutralize the negative charge investigations into membrane stability, cell morphology, chemotaxic of the phosphate group in the lipids, thus leading to a positively charged -membrane which then interacts electrostatically with actin. Our results responses, and endocytosis. Taken together, these data suggest that suggest that two types of actin-membrane attachments may exist in cells, ponticulin is responsible for the stable high-affinity linkage between the direct electrostatic interaction with lipids and anchoring via intrinsic Dictyostelium plasma membrane and the actin based cytoskeleton. proteins. Surprisingly, the absence of this interaction does not seem to decrease cell viability under normal laboratory conditions. Monday. Cytoskeleton-Membrane Interactions II (981-986) 169a

981 982 MECHANICAL LINKAGE BETWEEN RED CELL SKELETON CHARACTERIZATION OF DROSOPHILA cDNAs OF THE PROTEIN 4.1 AND PLASMA MEMBRANE IS NOT A DEMONSTRATED SUPERFAMILY ((M.Parra, M. Pallazolo, N. Mohandas, and J.G. Conboy)) FUNCTION OF PROTEIN 4.1. ((D. Discher, D. Knowles, S. McGee, J. Life Sciences Division, Lawrence Berkeley Laboratory, University of Chasis, M. Pafrs, J. Conboy, N. Mohandas.)) Lawrence Berkeley Laboratory, U.C. Beakeley, Bakeley CA. California, Berkeley, CA. (Spon. by J.A. Chasis) Protein 4.1 has generally been considered to have a dual role in red cell The prototypical 8OkD band 4.1 is a structural protein of the erythrocyte membrane physiology. It cerainly stabilizes -actin in the skeleton, but it membrane skeleton. In has also been thought to link the red cell skeleton to the bilayer through differentiating erythrblasts, splicing-mediated insertion its a 21 aa cassette in an to binding to Band 3, Glycophorins A and C, and phosphotidylserine.The best of internal -l0kD peptide imparts 4.1 a spectrin-actin evidence that 4.1 might be a mechanically-important link between the skeleton crosslinking activity, thus stabilizing the membrane against fragmentation by and bilayer had come from a fluid shear fragmentation assay of Gly C(-) external shear forces. 4.1 also may link the skeleton to the overlying bilayer by membranes (100% deficient). These membranes were found to fragment more binding to integral membrane proteins via an N-terminal domain homologous to readily than nornal. Recently, however, both 4.1 and a recombinant protein ezrin, moesin, and radixin. As a first step toward exploring the role of the 4.1- consisting of just the spectrin-actin binding domain of 4.1 (Glu400 to Gly486) like gene family in nonerythroid tissue in a genetically tractable organism, we were put into the GlyC(-) membranes by reversible hemolysis with the result that have initiated a search for Drosophila 4.1-like proteins using RT/PCR membrane strength was normalized. Evidence from membrane binding studies techniques. Two sets of degenerate oligonucleotides, complementary to the and quantitative eletrophoresis indicate that GlyC(-) membranes actually have a region of shared homology among all members of the 4.1 superfamily, were minor deficiency in 4.1. Most importantly, it is the missing complement of 4.1's used to cDNA. The resultant -150 nt PCR were spectrin-actin stabilization which weakens the GlyC(-) membrane. amplify Drosophila fragments Indeed, subcloned and sequenced. From one set of primers, two clones previous results show that easily fragmentable membranes with -40% deficit in independent were obtained whose deduced amino sequences in were 4.1 can be made normal in fragmentability by addition of just the spectrin-actin acid this region 43% binding domain (JBC 268:7186). Additionally, FRAP measurements on mAb and 50% identical, respectively, to human protein 4.1. This degree of labeled nonnal cells indicate that at least one-half of GlyC molecules are mobile. homology is similar to that observed earlier between mammalian and Drosophila Moreover, the immobile fraction was identical in both deformable and non- spectrin cDNAs. The second set of primers generated a single cDNA type with deformable cells, suggesting independence of the skeleton. Finally, preliminary striking homology to human radixin (80% identity over 46 amino acids). All results indicate low-affinity membrane binding for the N-terminal 30 kD domain three cDNA fragments hybridized to single bands on Southern blots of of 4.1, a domain with high homnology to portions of ezrin, moesin, radixin, and Drosophila genomic DNA, indicating that each is encoded by a single copy talin. Although this interaction may potentiate a mechanical coupling between gene. Our results suggest that Drosophila expresses multiple genes of the skeleton and bilayer, we must conclude that 4.1's primary role in membrane protein 4.1 superfamily. Utilization of Drosophila genetics to map and strength arises from its stabilization of the spectrin-actin network. manipulate expression of these genes may shed light on the functional role protein 4.1-like molecules may play in nonerythroid tissues.

983 984 MEMBRANE ASSOCIATION OF HUMAN P55 IN A NOVEL COMPLEX ROLE OF DEPHOSPHORYLATION IN MEDIATING THE ASSOCI- WITH PROTEIN 4.1 AND GLYCOPHORIN C. ((S.M. Marfatia', R.A. Lue?, D. ATION OF PROTEIN 4. 1 WITH THE CYTOSKELETAL STRUCTURE Branton2, and A.H.Chishti')) 'Department of Biomedical Research, St. OF HUMAN PLATELETS. ((T Mtsuoka ,M.Nishikawa Elizabeth's Toyoda2 and R.Yatani )) Department of Pathology and Medical Center, Tufts University School of Medicine, Boston, MA 02135; 2nd Department of Internal Medicine,Mie University 2Department of Cellular and Developmental Biology, Biological laboratories, School of Medicine,Tsu,MieSl4,JAPAN. (Spon. by Harvard University, Cambridge, MA 02138. (Spon. by S.-C. Liu.) M. Iwamoto) Protein 4. 1 is one of the three major structural P55 was first identified as a 55 kDa protein that copurified with dematin, an actin- proteins of the erythrocyte membrane skeleton, along bundling phosphoprotein associated with the human erythroid membrane skeleton. with spectrin and actin. When bound to the actin- P55 is a palmitoylated peripheral membrane phosphoprotein whose primary binding protein spectrin, protein 4.1 promotes the structure includes a single copy of the src homology 3 (SH3) motif, a C-terminal formation of stable ternary complexes with spectrin and F-actin. Phosphorylation of protein 4.1 results guanylate kinase domain, and an N-terminal domain of unknown function. In this in decrease in its affinity for spectrin and in its report we provide evidence for the direct association of p55 with the N-terminal 30 ability to promote spectrin-actin association, In kDa domain of protein 4.1, a key component of the erythroid membrane skeleton. vitro. Since spectrin and protein 4.1 are present in In addition, p55 also binds to the cytoplasmic domain of glycophorin C, a platelets, we used human platelets as a model to elucidate the role of phosphorylation of protein 4.1 transmembrane protein of red blood cells. We also provide evidence demonstrating in membrane skeleton-cytoskeletal interaction. Here the direct association of the N-terminal 30 kDa domain of protein 4.1 with the we show that, in unstimulated platelets, protein 4.1 cytoplasmic domain of glycophorin C. Taken together, these results suggest the is phosphorylated, and spectrin and protein 4.1 existence of a novel ternary complex at the erythroid plasma membrane involving remain in Triton-soluble fraction from the platelets after centrifugation. In platelets activated by p55, protein 4.1 and glycophorin C. The interaction of protein 4.1 with thrombin, phorbol ester, or stable thromboxane A2, glycophorin C is known to play a critical role in the regulation of membrane the rate and extent of protein 4. 1 dephosphorylation mechanical properties and erythroid shape. Since isoforms of p55, protein 4.1 and paralleled the association of spectrin and protein glycophorin C are present in many non-erythroid cells, the reported results describe 4.1 with the Triton-insoluble cytoskeletons. These results indicate that phosphorylation a yet unrecognized membrane-cytoskeleton of broad significance. reaction of linkage protein 4. 1 may be the basis for a mechanism that regulates membrane skeleton-cytoskeletal interaction in platelets.

985 986 REORGANIZATION OF THE RED IN STABLE CHANGES IN THE ERYTHROCYTES CYTOSKELETON. ((T. Ahmad, DEFORMATION ((D. Discher, A. Leung, N. Mohandas, E.A. Evans.)) Lawrence M. Mezna, S. Chettibi and A.J. Lawrence)) Department of Cell Berkeley Laboratory, U.C. Berkeley, Berkeley, CA, and U.BritColumbia, Vancouver, Biology, The University of Glasgow, Glasgow G12 8QQ, U.K.

The red cell's ability to deform is well known, but a major unanswered Free long-chain fatty acids enter the erythrocyte membrane and question is how the membrane components reorganize in deformation. To produce a characteristic pattern of membrane crenation seen by begin examination of the molecular responses we are quantitating fluorescence scanning EM. This treatment sensitises the cells to the lytic maps of membrane components in large cell deformation. Thus far, we have action of venom phospholipase A2 enzymes, but the response is observed two classes of response which are most readily described as those of extremely sensitive to inhibition by endogenous lysophospho- either a surface fluid or a surface solid. Optically-smooth, axisymmetric lipids. This inhibition has the unexpected feature that lyso- membrane deformations are produced by aspiration of osmotically-pressurized phospholipids are more inhibitory when added before the fatty red cells or ghosts into -2 ±m pipets. Density maps of fluorescing lipid and acid than after it. Extraction of free fatty acid from the protein in the aspirated tongue of membrane are then quantitated by CCD membrane by albumin does not reverse crenation as predicted photometry. The lipid bilayer, when labeled with highly mobile, lipophillic from the bilayercouple model and it greatly increases probes, has a uniform density along the membrane tongue. This is the sensitisation of the cells to the lytic action of phospholi- response expected for a homogeneous surface liquid in static equilibrium. In pase A2, but not to direct lytic agents. Inhibition of the contrast, the red cell skeleton, when labelled with either phalloidin or anti- action of fatty acid by lysophospholipids is also preserved spectrin Fab, exhibits a strong but smooth gradient in density: network dilation by albumin extraction, but the effects of addition order are occurs at the tip of the membrane tongue while network comprssion is evident reversed, lysophospholipids being much more effective when at the pipet entrance. This is the response expected for a compliant network; added before than after fatty acid. The model proposed is that intercalation of fatty indeed, this solid-like response appears stable for at least a half-hour, acids into the outer lamella of indicating that the overall structure is not very labile even when stressed. The the membrane changes the organisation of the spectrin skeleton and that the changes are same density map is also found for transmembrane receptors, probably stable to subsequent removal of indicating receptor localisation by the defonned network. However, in contrast exogenous lipid species. Models to interpret the effects of to the transmembrane receptors which have cytoplasmic domains capable of lipid addition order are based on the hypothesis that the network interaction, the GPI-linked receptor CD-59 concentrates at the tip of changes are associated with the mechanism of entry of the the membrane tongue. Steric exclusion by the extrafacial component of the fatty acid and not with its presence within the membrane. transmembrane receptors may drive this complementary, fluid-like response. Fluorescence-coupled micromanipulation is thus providing fairly detailed maps of the membrane emoganization which can occur in red cell deformation. 170a Cytoskeleton-Membrane Interactions II (987-992). Monday

987 988

CR3 (INTEGRIN OAB2) EXPRESSED BY TRANSFECTED K562 CELLS THE SMALL GTP-BINDING PROTEIN RAC REGULATES LAMELLIPODIAL AND COOPERATES WITH FCRII FOR PHAGOCYTOSIS OF IGG-OPSONIZED FILIPODIAL DYNAMICS AND CONTROLS CELL-SUBSTRATE ADHESION IN XTC PARTICLES.(II.L. Graham, V.M. Holers, and E.J. Brown)) Departments of FIBROBLASTS. ((M. Symons1, R.-G. Qiu1, L. Conroy2, R. Clark1, Pediatrics and Internal Medicine, Washington University School of Medicine, T. Mitchison3 and F. McCormick1)) lOnyx Pharmaceuticals, St. Louis, MO 63110. Richmond, California, 2Cetus Corporation, Emeryville, California and 3Department of Pharmacology, University of Leukocyte B2 integrins have a role in the phagocytosis of IgG-opsonized California, San Francisco, California. particles by neutrophils and monocytes, especially when ingestion has been We have previously obtained evidence for the existence of a stimulated to a maximal level with phorbol esters. Antibody data suggest ras-like GTP-binding protein involved in the control of actin that CR3 is particularly important for this function. In order to study the cytoskeleton and cell-substrate adhesion using microinjection functional interaction between IgG Fc receptors and CR3, we have stably of guanine nucleotide analogs in Xenopus XTC fibroblasts (J. transfected K562 cells, which constituitively express FcRII but not other IgG Cell Biol. =L , 1235). We therefore investigated the role of receptors or any and cDNAs. In the absence of Fc 112 integrins, with am 12 rac, a GTP-binding protein involved in the regulation of plasma CR3, K562 cells bind IgG-opsonized particles, but show minimal ingestion membrane ruffling. Injection of the constitutively active either with or without phorbol esters (Pi - 5-10). The CR3 transfected cells mutant rac2L61 in XTC fibroblasts inhibited filipodia and bind complement opsonized particles avidly, but phagocytose these only at ruffling, induced the formation of lamellipodia and increased a low rate following phorbol ester treatment (Pi -20). Particles opsonized cell spreading. No alterations in stress fibers could be with both IgG and complement are ingested much more efficiently (PI =130), detected. Injection of a dominant negative rac mutant had opposite effects: it inhibited lamellipodia and cell spreading demonstrating synergism between CR3 and FcRII for ingestion. Importantly, and stimulated formation of filipodia. These changes in cell while the CR3 transfectants bind particles opsonized with IgG alone morphology are independent of rho function, a GTP-binding equivalently to controls, phagocytosis of these targets following phorbol protein which controls the formation of stress fibers, since ester stimulation is enhanced 4-10 fold, in the absence of any known CR3 pre-injection of C3 transferase did not inhibit the changes in ligand. Target particles opsonized with either fibronectin or anti-HLA mAb are cell morphology induced by rac2L61, although it strongly minimally ingested by either transfectants or control cells with or without diminished stress fiber formation. Injection of rac2L61 also phorbol esters. These data demonstrate a PKC-dependent interaction strongly inhibited cell rounding caused by GRGDSP peptides but between CR3 and FcRII which markedly enhancesIgG-mediated phagocytosis had no effect on cell rounding caused by trypsin, indicating that activation of rac leads to an increase in cell-substrate at a step subsequent to particle binding. This transfection system will allow adhesion. The effects of the various rac mutants suggest that a detailed biochemical analysis of receptor cooperation in phagocytosis. rac may regulate lamellipodial and filipodial dynamics via its control of cell-substrate adhesion.

989 990 DYNAMIC MEASUREMENTS OF INTEGRIN INTERACTIONS DEREGULATED pp60O TRIGGERS CONTRACTION OF THE CORTICAL WITH THE LYMPHOCYTE CYTOSKELETON. ((D.F. Kucik, and CYTOSKELETONINXENOPUSOOCYTES. ((C.A. Larabell', T.F. Unger', and R.E. Steele')) 'Life Sciences Div., Lawrence Berkeley Laboratory, Berkeley, CA E.J. Brown)) Department of Intemal Medicine, Washington University 94720 and+Dept. of Biological Chemistry, Univ. of California,Irvine, CA 92717. Medical School, St. Louis, MO 63110 Previous studies showed that Xenopus pp60O with constitutive kinase activity Lymphocyte adhesion to ICAM-1, the ligand for integrin aL02 is triggers morphological changes in the Xenopus laeis oocyte cortex, including increased by treatment with phorbol esters, without any change in aggregation of pigment granules and apparent invagination of the cell surface (Unger and Steele, Mol. & Cell. Biol. 12:5485-5498, 1992). In this study we surface density of the integrin. We have used the technique of single use electron microscopy and immunocytochemistry to further investigate these to test hypothesis that changes in cytoskeletal particle tracking (SPT) the changes. Injection of RNA encoding the deregulated variant of Xenopus pp60- interactions of aL2 are involved in this phenomenon. Gold particles results in formation of a denseband of filaments just beneath the plasma membrane coated with mAbIB4 (specific for J2) were allowed to settle onto EBV- around the injection site. The membrane in this region is more highly folded and transformed B cells, and their motion was observed with video-enhanced the cortical organelles are crowded together, suggesting a contraction around the site. This contractile phenomenon next DIC microscopy and analyzed by computer. At baseline,IB4-coated injection continues over the several hours gradually encompassing a larger region of the oocyte cortex. While previous particles show directed centripetal movement, but little random motion, studies suggested that the cortex eventually invaginates, electron microscopy consistent with cytoskeletal attachment. Phorbol ester treatment causes reveals that this isnotthecase. Instead, the band offilamentsjustbeneaththe the motion of (2 integrins on the dorsal surface of the lamella to be less plasma membrane contracts so strongly that it detaches from the plasma constrained, consistent with release from the cytoskeleton. This is in membrane, severing the attachments of the cortical organelles to the cell surface. The organelles gradually sink deep into the internal . The point of contrast to complement receptor 1, whose baseline cytoskeletal interac- detachment of the dense band of filaments from the plasma membrane can be seen tions are markedly different from those of the (2 integrin, and don't at the periphery of the affected area. The contractile band extends across a large change with phorbol ester treatment. Release of (2 from the cytoskele- region of the oocyte around the injection site and is located as much as 10 jum (at ton occurs with similar kinetics to increased adhesion of cells to ICAM- 8 hr) beneath the plasma membrane. The cortical and pigment granules are 1. These data suggest a model in which phorbol ester mediated situated even more deeply in the cytoplasm. Immunogold analyses indicate the presence of actin filaments in this band. These studies that, in Xenopus release of this integrin from the cytoskeleton is important in regulation suUest oocytes, deregulated pp60(" triggers phosphorylation of a protein capable of of adhesion. Experiments are underway with mutant (2 transfected into activating an actin-containing contractile network in the oocyte cortex. Supported 32-deficient lymphocytes to test this model. by N.I.H. grant RR05097 (IVEM Fac. LBL) and N.S.F. grant IBN-9204907 (RS).

991 992

A NOVEL PROTEIN-TYROSINE PHOSPHATASE HAS HOMOLOGIES TO BOTH MAPPING THE HETERO- AND HOMOTYPIC ASSOCIATION DOMAINS OF THE CYTOSKELETAL PROTEINS EZRIN AND JUNCTION-ASSOCIATED EZRIN AND MOESIN. ((R. Gary and A. Bretscher)) Section of GUANYLATE KINASES. ((K. M. Walton and J. E. Dixon)) Dept. of Biol. Chem., Univ. Biochemistry, Molecular and Cell Biology, Comell University, Ithaca, NY of Mich., AnnArbor, Ml 48109. 14853.

The networkis involved In the control of shape, migration cytoskeletal cellular Ezrin and moesin are closely related (74% sequenceidentity) andinteraction wIth the extracelkilar environment. In fbroblasts, talin connects actin putative membrane-cytoskeletal linking proteins found In surface filaments to the membrane throughits association with vinculin and theintegrin structures of many cells In cultures and In tissues. Although specific receptor. The erythrocyte protein band 4.1 binds to spectrin and actin, anchoring membrane and cytoskeletal binding partners of ezrin and moesin remain them to the membrane viaits associton with glyophorin. Band 4.1 andtaHin areIn a to be of truncated forms of ezrin has shown famly of related cytoskeletal protelns thatIncljdesmoesin,ezrin, and radixin. The Identified, transfection that redistribution of these and other cytoskeletal proteins during cellular processes the N-terminal half of ezrin (residues 1-309)is responsible for plasma correlates with changesIn their tyrosyl-phosphorylation state. In addition, the membrane localization of the protein, and the C-terminal half (residues integrity ofjunctional complexes may be regulated by tyrosine phosphorylation. 280-585)colocalizes with the actin cytoskeleton (Algrain et al. JCB it Thus, isNlkely that proteintyrosine phosphatases (PTPs) have a central role in 120:129, 1993). Ezrin and moesin bind to one anotherin vivo andin regulating cytoskeletal activities. vitro, and each proteinIs also capable of self-association as determined To obtain novel PTPs which may beinvolvedin these processes, we probed the by a blot overlay binding assay (Gary and Bretscher, PNAS, In press). To neural crest-derived adrenal medulla by PCR directed toward two conserved investigate the moiecular organization of these proteins, we used chemical sequence motifs wIthin PTP ofknown phosphatases. Eighteen the domein differert cleavage at cysteine residues with DTNBfoilowed by blot overlay of the were cloned: six represented novel phosphatases. We pursued one of the PTPs partial digestion fragments to map the hetero- and homotypic association novel PTPs that did not show strongsimiarIty to any known PTP. This phosphatase, domains of each protein. The cysteine cleavage fragments wereidentified BA14, has an mRNA size of approxImately 8 kb. The enzymatic activity of the on the basis of molecular weight, and theidentitiesconfirmed by the phosphatase domainis specific for phosphotyrosine and has no activity against agreement between and of phosphoserine. Analysis of the derived protein sequence indicates that BA14 has observed predictedincorporation [14C]-KCN one domain with similarity to the amino-terminal hal of the ezrin-moesin-radixin Into C-terminal but not N-terminal cleavage products. The fragments were a protein family. In addition, BA14 has five repeats of the'GLGF moti, identified in electrophoresed, transferred to blot, and probed with blotinylated both the Drosphla tumor suppresser dig, a guanylate kinase localized to septate ezrin and moesin. Fragments to which the biotinylated proteins had bound lunctlons, ard the protein PSD-95, a guanylate kinase localized topostsynaptic wereidentified using avidin-peroxidase to locate the blotin tag. For denskItes. The presence of these motifs strongly suggest that BA14 localizes to the ezrin, both heterotypk andhomotypic binding activities werecontalned cytoskeleton, and mey regulate the function of junctional complexes. Supported in within the region from Cys-283 to Leu-585, and for moesin the region part by gratts from the NIH. from Cys-283 to Met-576 was responsible for both types of binding. Monday. Cytoskeleton-Membrane Interactions II (993-998) 171a

993 994 ANTAGONISTIC CHEMORECEPTORS MOBILIZE ACTIN INTO GROWTH CONE ENRICHMENT AND CYTOSKELETAL AND OUT OF ANEMONE HAIR BUNDLES. ((G.M. Watson and J. ASSOCIATION OF NON-RECEPTOR TYROSINE KINASES. ((S.M. Roberts)) Dept. of Biology, Univ. Southwestern Louisiana, Lafayette, LA 70504. Helmke and K.H. Pfenninger)) Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, CO 80262. Hair bundles on sea anemone tentacles regulate discharge of nematocysts into swimming prey. Fetal rat brain (E18) expresses four Src-like membrane-associated non- Activating surface chemoreceptors for N-acetylated receptor tyrosine kinases: Src, Fyn, Lyn, and Yes. Src and Fyn are the sugars induces hair bundles to elongate. Subsequently most abundant and are enriched in a subcellular fraction of nerve activating chemoreceptors for induces hair highly cones bundles to shorten. Previously, we found that growth (GCPs). To study the cytoskeletal association of these activating chemoreceptors for N-acetylated sugars tyrosine kinases, Triton X-100-resistant fractions are prepared from GCPs. caused a 50% increase in fluorescence intensity of All four non-receptor tyrosine kinases are associated with the cytoskeleton hair bundles stained with phalloidin-rhodamine. to a significant degree. The associations are sensitive to ionic strength, Subsequently activating receptors for proline and the relative affinities are Fyn > Src > Lyn > Yes. To investigate decreased fluorescence intensity to control levels. the regulation of the Src association we used phosphatase inhibitors to We now find evidence for a pool of G-actin located increase in beneath the stereocilia as indicated by punctate phosphotyrosine levels GCPs. This results in the release of Src DNase-I-rhodamine labeling. While activating from the cytoskeleton. The released Src is highly phosphorylated on tyr receptors for N-acetylated sugars caused a 37% 527. These findings are explained by the following: Src associates with decrease in DNase-I labeling (a decrease in the the cytoskeleton via its SH2 domain; it is released when tyr 527 in its density of points of light), subsequently activating regulatory domain is phosphorylated and binds intramolecularly to the SH2 proline receptors restored DNase labeling to 91% of domain. The control values. These data suggest. that activated cytoskeletal association of the other src-like tyrosine kinases to receptors for N-acetylated sugars mobilize G-actin is likely be regulated similarly since these domains are highly into the stereocilia where it polymerizes during conserved. Our studies indicate that Src family members are directly bundle elongation. Activated receptors for proline involved in the mechanism of regulated membrane-cytoskeleton interaction mobilize actin out of these stereocilia, inducing in normal cells. (Supported by NSF grant BNS 9109775) bundles to shorten and restoring the G-actin pool. Supported by NSF DCB-9105058.

995 996 ATTENUATION OF F-AcTIN FLow OccURS ALONG THE Axis OF MICROTU8ULE THE LOCALIZATION OF ERM PROTEINS IN GROWTH CONES IS DISRUPTED BY ExTENSION DURING GRowTH CONE-TARGET INTERAcTIONS ((C.-H. Lin and P. GROWTH CONE COLLAPSE ((Ch. Gonzalez Agosti and F. Solomon)) Department of Forscher)) Departnent of Biolgy, Yale University, New Haven Connecticut Biology and Center for Cancer Research, M.I.T., Cambridge, MA 02139 06511. The monoclonal antibody 13H9 stains the growth cones of several neuronal cell types. The localization of the 13H9 antigen is similar, though not to that of and is In a recent report we provided evidence that biomechanical forces associated identical F-actin, clearly distinct from that of microtubules. However, the loalization of the 13119 antigen to with reograde F-actin flow are applied to targets during physiological neuronal growth cones depends upon intact microtubules. Exposure of neurons to nocodazole induces growth cone-target interacto. During such iriteractions, coupling occurs both microtubule depolymerization and concomitant debocalization of the antigen from beween growth cones and target subatrates accormanied by directed extension growth cones (Goslin etat., JCB 109:1621, (1989)). In contrast, localization of the 13H9 of microtubules and the central cytoplasmic domain toward the ske of target antigen to theposition of the marginal band of chicken red blood cells is completely contact. These directed microtbule movements were randomized by independent of the stat of microtubules (Birgbauer and Solomon, JCB 109: 1609 (1989)). cytochalasin treatment, suggesting microfilament-microtubule interactions. (Un We have mapped the epitope recognized by Mab 13H9; a hexapeptide contained in the and Forscher (1993) J. Cel Biol. 121:1369). In this study we further investigated sequence of aU members of the ERM-family (ezrin, radixin, moesin) as wen as the more the relationship between F-actin flow and target coupling. To do so, a spatial distantly related protein merlin is sufficient for binding of Mab 13119. To study the role of these proteins in motility, we have pertubed growth cone behavior. map of F-actin flow dynamics was obtained by strategically placing markers on Using chick embryo sympathetic neurons, we followed the consequences of growth cone collapse the growth cone surface using a beam gradient force optical particle trap (laser for the 13H9 staining pattern In these cells, growth cones collapse upon withdrawal of NGF tweezers) during target interactions. Polstyrene microbeads known to couple or upon contact between two growth cones. We found that the 13119 antigen was delocalized were as passively to the retorade F-actin flow utilized markers to monitor flow from the tip of the neurite concomitant with the collapse of the growth cone induced by either rates in regions of interest. Beads placed within a corridor along the interaction mechanism. After two hours without NGF, the 13119 staining was undetectable at the tips. At axis exhbited a 2-5 fold attenuation in retrograde flow rates and sometimes short times afterreaddition of NGF(2 to 10 min), the 13119 staining reappeared at the tips stopped completely or reversed direction, moving toward the distal contact site. with the sametime course as reappearance of the growth cone lmelipodia. Using double In marked contrast, beads placed off of the interaction axis continued retrograde immunoflrce, we compared this behavior to that of microtubules and F-actin in the translocation at control rates. Our results suggest: 1) a reciprocal relationsh4 distal neurite. Ourresults are consistent with an important role for 13H9 andgen in the between F-actin flow and central (microtubule) cytoplasmic domain extension organization of the growth cone. rates and 2) generation of tension in the actin cytoskeleton along the interaction axis may be aphysical mechanism for target site directed microtubule, and thus axonal, guidance. (Supported by: NIH NS28695 & The McKnight Endowment Fund for Neuroscince)

997 998 INTRACELLULAR DELIVERY OF anti-GAP43 ANTIBODIES INTO STRUCTURE AND FUNCTION OF DROSOPHILA SPECTRIN'S NEUROBLASTOMA INHIBITS NEURITE OUTGROWTH INDUCED BY SERUM HEAD-END INTERCHAIN BINDING SITES. ((H. Deng, J. Lee, and D. DEPRIVATION MORE THAN OUTGROWTH ELICITED BY dbcAMP TREATMENT Branton)) Departments of Biochemistry and Molecular Biology and ((T.B. Shea, M.L. Beermann)) McLean Hospital, Harvard Medical School, Belmont Cellular and Developmental Biology,Harvard University, Cambridge, MA MA (Spon. by 1. cher) 02138. The growth-associated protein GAP43 (also referred to as B50, Fl, pp46 and neuromodulin)is expressed In large amounts during devewpment and regeneration The association between a- and fspectrin at the "head end" of the spectrin ofneural connections, where itis prominentin growth cones and along the axonal molecule gives rise to tetramers and is essential for the integrity of spectrin length. Conflctng evidence obtained ina variety of modelsystems has generated based membrane skeletal networks. We have analyzed the head-end associ- controversy regarding whether or not GAP-43 plays an essential role In neurite ations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation outgrowth. Our previous sudies have demonstrated that thehntracellular delivery of assays using protein fragments synthesized in vitro from recombinant DNA anti-GAP43 antibodies inhibis the initiation of axonal neurite outgrowth in NB2a/d1 neuroblastoma. Related studles from this laboratory have demonstrated that showed that the head-end binding sites reside exclusively within a-spectrin whle segments 0-1 and segment 18. No neurites elaborated following serum deprivation or dbcAMP treatment (in the ,B-spectrin specific interchain binding presence of serum) are both dependent upon microtubule polymerization and sites were found in a-spectrin segments 3-17 or f-spectrin segments 5-17. substrate adhesiveness, neurates elaborated In response to serum deprivation are The head end interchain binding sites of Drosophila spectrin associate with more dependent upon substrate adhesiveness than are those elaborated by an affinity and temperature dependence similar to those of erythroid dbcAMP treatment. In the present study, we compared the effect of intracellular spectrin. Polypeptides containing the Drosophila binding sites could delivery of various concentrations of anti-GAP43 antibody on neurite outgrowth compete with the erythroid spectrin interchain binding sites. Several point elcted by serum deprivation with that elicited by dbcAMP treatment. Serum mutations equivalent to erythroid spectrin mutations responsible for deprivation and dbcAMP treatment In the presence of serum for 4hr Induced neurite hemolytic anemias in humans also abolished head-end interchain binding in elaborationinsimilar percentage ofcells (e.g, 42.1 and 51.5, respectively). While Drosophila spectrin. To test the importance of spectrin networks in vivo, Intracellular delivery of suffclent antisera concentrationsinhibitedneurite outgrowth we attempted to rescue Drosophila mutants lacking a functional a-spectrin by treatments, an series that as as a both antibody dgution revealed much 50% gene by transforming them with minigene constructs carrying wild type or Inhibition of neurite elaboration by serum-deprivation could be attained in the head-end interchain binding deficient spectrin. A range of absence of anyInhibtion of dbcAMP-induced neurite outgrowth. A provisional phenotypes were condusion from these studiesi that the role of GAP-43in neurite outgrowth may observed. The specific phenotype depended on the nature of the interchain involve modulation of critical aspects of membrane adhesiveness. One prediction binding deficiency that had been introduced and, remarkably, on tempera- arisng from these results would be that cuituring of neuronal cells on asutfdlently ture. adhesive substrate, and/ or under conditions that maximize microtubule polymerization, may lesen or supplant any requirement for GAP-43. Suppored by NSF BNS-s010981. 172a Cytoskeleton-Membrane Interactions II (999-1002). Monday

999 1000 NEURONAL COMPARTMENTALIZATION OF BRAIN B SPECTRIN NEURONAL COMPARTMENTALIZATION DEMONSTRATES ISOFORMS ((Y.Ma', M.B. Clark', M.L. Bloom2, J.E. Barker2, I.S. DISTINCT a SUBUNITS OF BRAIN SPECTRIN ISOFORMS ((M.B. Zagon3, W.E. Zimmer', S.R. Goodman1)) 'Department of Structural and Clark and S.R. Goodman)) Department of Structural and Celhslar Cellular Biology, University of South Alabama College of Medicine, Biology, University of South Alabama College of Medicine, Mobile, AL Mobile, AL 36688; 2Jackson Laboratories, Bar Harbor, ME 04609; 36688. 3Department of Neuroscience and Anatomy, M.S. Hershey Medical Center, 17033. The availability of isoform and subunit specific antibodies (Riederer et al, 1988, Br. Res. Bull. 21: 607-616) has allowed us to determine the The recent completion of the sequence of mouse BSpIIE (Ma et al, Mol. immunohistochemical localization of a subunits of brain spectrin isoforms Br. Res. 18: 87-99, 1993) and mouse BSpIEl (Bloom et al, Blood in press), and compare mese results to the localization of the B subunits of these by our laboratories, has allowed the preparation of peptide specific isoforms (Ma et al, this meeting). The specificity of the antibodies for antibodies to unique sequences within these B spectrin isoforms. A 15-mer isoform specific ot subunits of mouse RBC spectrin and brain spectrin was representing residues 6-20 of mouse BSpIE 1, and its alternately spliced determined by quantitative immunodot and immunoblot methods. form BSpIE2 found in brain, was used to prepare an antibody which reacts Immunohistochemical analysis revealed that antibodies specific for the ax exclusively with erythroid B spectrin on Western blots and quantitative subunit of erythroid spectrin (ciSpIEl) localized antigen to the somata and immunodots. A 15-mer representing residues 2139-2153 of mouse BSplIEl dendrites of cerebellar granule cell neurons, a pattern identical to that was used to prepare an antibody which reacted exclusively with seen for localization of the erythroid B subunit (BSpIE2) (Ma et al, this nonerythroid spectrin. Immunohistochemical analyses indicated that meeting). In contrast antibodies specific for nonerythroid a subunit BSpIE2 is located in neuronal somata and dendrites in mouse cerebellum, (aSpIlE1) localize antigen to in the cerebellum corresponding to the and therefore represents the subunit of brain spectrin (240/235E) pattern for the nonerythroid B subunit (BSpIlE 1). The distinct localization (Riederer et al, J. Cell Biol. 102: 2088-2097, 1986). BSpIlE is located in of antigens by antisera which recognize either the ax subunit of RBC axons and soma of neurons, and therefore represents the subunit of spectrin or theca subunit of nonerythroid brain spectrin together with te brain spectrin (240/235) (Riederer et al, 1986). These peptide specific B correspondence of their localization with appropriate B subunits clearly spectrin antibodies represent the most exactng current reagents for indicate that brain spectrin consists of two isoforms each with disnct spectrin immunohistochemistry and immunobiochemistry. and B subunits, namely, erythroid brain spectrin, ctSpIEl/BSpIE2, and nonerythroid brain spectrin ctSpIIEl/BSpllE1.

1001 1002 ASSOCIATION OF A SPECTRIN ISOFORM WITH THE GOL(GI MICROINJECTION OF SPECTRIN ANTIBODIES INDUCES CLEAVAGE APPARATUS ((K. A. Beck and W. J. Nelson)) Dept. Molecular and DELAY AND SURFACE INSTABILITY IN STARFISH EMBRYOS. ((G. Cellular Physiology, Stanford University School of Medicine, Stanford, K. Wong, D.H.R. Hoyle, and D.A. Begg)) Department of Anatomy and Cell CA94305-5426 Biology, University of Alberta, Edmonton, Alberta T6G 2H7

A number of studies from our lab and others have indicated that the spectrin (fodrin) We have used antibody microinjection to study the role of spectrin in cell based membrane skeleton participates in the formation of membrane domains in division and early development. Affinity-purified polyclonal antibodies to sea polarized celis. We have raised an antibody to the p subunit of canine erythrocyte urchin egg spectrin was mnicroinjected into one blastomere of a two-cell stage spectrin and found that it reacts with a 220-kDa spectrin homologue in a variety of starfish embryo (Pateria miniata). The uninjected blastomere served as a non-erythroid cells including the epitheial cell line MDCK. Immunofluorescence control. Injection of 0.2-1 ng of antibody delayed the onset of cleavage and experiments have revealedthat this spectrin isofoem, unlike fodrin, is localized to the increased the occurence of surface blebbing following cytokdnesis. Cleavage Golgi appats of these cells. Golgi localization of spectrin was confirmed by delay resulted from a lengthening of the non-mitotic portion of the cell cycle. microinjection of canine spectrin into living MDCK rhodamine-labeled erythrocyte The time required for mitosis and cytokinesis itself was not altered. The which resulted in anacctnulation of spectrin on punctate peri-nuckar structures cells length of the cleavage delay increased as development proceeded, until the that co-localize with Golgi markers. A dynamic association of spectrin with the progeny of the injected cell ceased cleaving. Cells injected with lower doses Golgi was revealed by studies employing two pharmacological perturbants of Golgi of antibody showed less of a delay and completed more divisions function nocodazole and brefeldin A (BFA). After 30 min at 37-C in the presence of cleavage nocodazole, the Golgi apparatus was found as small fragments dispersed throughout than those injected with higher doses. Upon cessation of cleavage, cells the cytoplasm, whereas spectrinexisted primarily as large peri-nuclear structures exhibited increased surface activity, extended motile protrusions, and reminiscent of steady state Golgi morphology and distribution. With longer frequently became amoeboid. The progeny of the injected cells did not incubations (>90 min) spectrin co-loalized with dispersed peripheral Golgi elements. participate in the formation of the blastula epithelium, but instead formed an Treatment of MDBK cells with BFA resulted in rapid (<2 min) release of spectrin aggregate of highly motile cells that were loosely associated with the from the Golgi that was coincident with the tubularization of the Golgi apparatus and epithelium originating from the uninjected blastomere. No significant effect correlated both temporally and spatially with the redistribution of the Golgi coat on cleavagetiming or cellmorphology was observed with control injections of protein p-COP. Ourresults show that an isoform of spectrin immunologicallyrelated buffer alone or purified preimmune serum. These results support the to erythroid spectrin is associated with the Golgi apparatus of non-erythroid cells hypothesis that spectrin participates in stabilizing the surface of blastomeres in where it may serve to function in the maintenance of steady state Golgi morphology developing embryos and in forming cell junctions. They also suggest that a and composition as well as in dynamic events that occur within this organelle in the normal pool alters including the sorting of newly synthesizedmembrane proteins. reduction spectrin cell cycletiming. Supported by Grants from the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada. Proteins (1003-1004)

1003 1004

THE STABILITIES OF NEUROFILAMENT MRNAS IN PRIMARY CULTURES TISaB CFIC ENaMEH w TM ornAiiir Mvy GM .( C. J. RAT DRG INCREASE DURING POSTNATAL DEVELOPMENT.((M.L. Schwartz, EicHom M V. (M.L.&*.mtt, Ratagi, Bru, and a)) of N of P.S. Shneidman, J. Bruce, and W.W. Schlaepfer)) Division of W.w.Shlaqspf Division lr-ttilogy,The tbivwuity PA Neuropathology, University of Pannsylvania Medical School, PFuoslvania MNlical Sc,l,Ehiladephia, 19104-6079. Philadelphia PA 19104 6079. b hav the E el nts r ctui4bl fr the prcadzsi pwtion of thenams rmsroila,tt hma gn (l-8) The ability of actinomycin (ACT) to prevent the loss of prasvK by la5 yj using attracts from uqs?ssdng three neurofilament (NF) mRNAs in primary cultures of adult (brain) and (liver) tissums. lf hav found that rat dorsal root ganglia (DRG) indicates that NF transcripts stna 5' region fram -1314 to -115 edibit a 3-5-fold are regulated posttranscriptionally and suggests that the NF higher levl of NF-H Pr -sntr activity, relative to the admirs transcripts are stabilized/destabilized by transcription- umior-late praster (pM), in brain vs livew attracts. DaletiAm to -85 dependent factors (Schwartzet al., J Biol Chem 267:24596, lanra the level of brain by abat 2- , ile deletio 1992). This study compares the loss of NF mRNAs in primary fro -65thbrugh -31 rae,..t shout 5-fold to a relatively DRG cultures from P2, P16 and P30 rats to determine whether bal l l 1/10 pjL). Basal level _ressio stabilization of NF mRNAs could account for the upregulation desrvad in all dalmtics t-cribed with liver attract. Delation to in NF expression during postnatal development (Schlaepfer and -24 (u-less) aboLid Wotar activity wdth both atrats. Block of -64 to -31 region vith ra su Bruce, J Neurosci Res 25:39, 1990). Whereas high levels of the nd mintained high leel brain no .un of iqmortssm in mRNAs are present in P30 (and adult) rat DRG for 24 hr In qresac this area. Daletion of the -115 to -65 region in a largr cautt vitro, losses of all three NF mRNAs occur in P2 cultures at rxelted in basal lwsl esprsion with brain attract suggesting that 8, 16 and 24 hr. Losses of NF mRNAs in P16 cultures occur this region contaim the slesnts rsKssay for th tissue times intermediate to those of P2 and P30 cultures. While ACT higlevel spacific prastar furcxtion. 39.tation of a seli une widthin not prevent the 0-24 hr loss of NF in P2 cultures, does mRNA this region reslted in reduction of activity to baal leveL. ACT-treated show low but levels of NF cultures similar mRNAs This lared activity correlatedwith the loss of a shifd btad gal - at 24 and 48 hr. These findings support the view that NF in shift assays.s=tAiUr SU Ft that (-106) _ st in transcripts in DRG neurons are stabilized by transcription- XTXTTXI(-72) is ispcLtA brain-pacfic high-level trvanpripion tim ter. independent factors whose activities increase in postnatal frXU NF-H pra.- development. The study also suggests the presence of transcription-dependent factors that destabilized NF mRNAs in developing and mature rat DRC neurons. Monday. Neurofilament Proteins (1005-1010) 173a

1005 1006 INFLUENCE OF REPORTER GENES ON ACTIVITY OF THE NF-L NF-M INDUCES PARALLEL BUNDLING OF INTERMEDIATE FILAMENTS AND PROMOTER: POSSIBLE INVOLVE1nNT OF MATRIX ATTACHMENT CROSSBRIDGES RESEMBLING THOSE IN THE . ((T. Nakagawa, J. REGIONS(MARs. ((G. Charron , J-P. Julien , and V. Chen, Y. Kanal, and N. Hirokawa))Department of Anatomy and Bibor-Hardy )) 1:Montreal General Hospital Research Cell Biology, School of Medicine, University of Tokyo, Tokyo, Institute, Centre for Research in Neuroscience, Japan(Spon. by K. Nagata) IcGill University, Montreal Quebec H3G 1A4. :Institut du cancer de Montreal, Montreal Quebec in nerve axons are arranged parallel to each H2L 4M1. other and form thick bundles as a core of axon. In addition frequent crossbridges are observed between them. Previous We tested the activity of the proximal promoter studies suggested that parts of NF-H and NF-M could be compo- region (-297 bp) of the human neurofilament light nents of these crossbridges. However, it was not clear how NF gene(hNF-L) in transgenic mice using three reporter triplet proteins form parallel bundles of neurofilaments. In genes,the chloramphenicol acetyl transferase(CAT), another words how individual neurofilament protein contributes the 5-galactosidase gene(lacZ) and the hNF-L gene in to form these characteristic neurofilament organization in which the intron have been deleted. Each fusion vivo. Because the function of NF-M was least unclear among the construct generated distinct expression patterns. triplet proteins as the first step toward this goal we ex- The hNF-L/CAT construct was expressed at low levels pressed NF-L or NF-L plus NF-M in Sf9 cells using baculovirus in various tissues of adult transgenic mice while infection system and examined the change of intermediate the hNF-L/LacZ construct was expressed exclusively filament organization. Although intermediate filaments in Sf9 in the during embryonic development. cells form loose network in cells expressing NF-L only, thick On the others hand, the intronless hNF-L transgene bundles are induced by expressing NF-L was expressed correctly in adult nervous tissue. and NF-M together. Both NF-L and NF-M were colocalized on Nuclear matrix binding analysis has revealed the these filaments. Furthermore the quick freeze, deep etch EM presence of a matrix-attachment region(MAR) within revealed parallely arranged intermediate filaments and fre- the 3'untranslate region of the 5-galactosidase gene quent crossbridges between them which quite resemble neurofi- and the hNF-L gene. Those regions are A/T rich and lament arrays in axons. This clearly indicated that NF-M bind to nuclear matrix in vitro and in vivo. Works actively induces neurofilament bundling and form crossbridges are now in progres to test, in transgenic mice, the between neurofilaments, thus plays an important role for possible role of these MAR sequences on the organization of neurofilaments in vivo. transcriptional activity of the hNF-L promoter.

1007 1008 ASSEMBLY PROPERTIES OF TRUNCATED NF-H PROTEINS WEIGHT NEUROFILAMENT PROTEIN, NF-H, LEADS TO ALTERATIONS IN THE WITH OTHER NEUROFILAMENTS IN THE PRESENCE AND STRUCTURE OF THE ENDOGENOUS NEUROFILAMENT NETWORKS OF CULTURED NEURONS. ((K.T. Straube-West, P. Opal, and R.D. Goldman)) Department ABSENCE OF . ((D. Sun, P. Macioce, S.S.M. Chin, of Cell, Molecular and Structural Biology, Northwestem University, Chicago, 1160611. and R.K.H. Liem)) Department of Pathology, Columbia University, New York, NY 10032. We have recently demonstrated that microinjected neurofilament triplet proteins (NFTs) are incorporated into the endogenous neurofilament networks of cultured chick dorsal root ganglion cells (DRGs) (Opal et al., Mol. Biol. Cell, 3S:355a, 1992). Furthermore, the rate In order to understand the roles played by the and pattern of incorporation are dependent on the type of neurofilament protein, with NF- subdomains of rat NF-H in coassembly with other H being incorporated almost twice as fast as NF-L at concentrations of I mg/ml in the injection buffer. This finding supports the idea that NF-H may have a more peripheral neurofilaments subunits and vimentin, we constructed location on the neurofilament than does NF-L. Two recent studies have demonstrated that amino- and carboxyl-teminal deletion mutants of NF-H overexpression of NF-L (Xu et al., Cell, 73:23, 1993) or NF-H (C6te et al., Cell, 73:35, and performed transient tranfections in and 1993) in transgenic mice can lead to the formation of neurofibrillary tangles in motor SW13cl1Vim+ neurons of these mice. We have directly addressed the importance of NFT stoichiometry SW13cl.2Vim- cell. Our results show that: 1) Both ends of in single cells by microinjecting higher (2-3 mg/ml) concentrations ofbiotinylated NF-H the NF-H rod domain are important for coassembly with into cultured chick embryo dorsal root ganglion cells. Within hours after microinjection, either vimentin or NF-L 2) The tail domain of NF-H is not the endogenous NFT network has pulled away from the periphery of the cell body and begun to collapse around the nucleus. Bundles of neurofilaments which enter the neurites important for coassembly with NF-L or with vimentin. 3) appear to remain intact. Within 24 hours, neurofibrillary tangle-like NFT networks are An amino-teminal rod deleted NF-H mutant is capable of observed which contain thick bundles of neurofilaments. These structures react with coassembly with NF-L only in the presence of vimentin. 4) monoclonal antibodies against ubiquitin and phosphorylated NF-H, antigens which are found in neurofibrillary tangles of motor neuron diseases such as ALS. Preliminary Full length NF-M coassembles only with NF-H protein observations suggest that there is also a decrease in neurite diameter. These results which has an intact rod domain in the presence of suggest that the balance of the three neurofilament subunits may be critical and that vimentin. 5) In SW13cl.lVim+ cells, a deletion of the alterations in the stoichiometry between the three subunits can lead directly to alterations in the endogenous neurofilament network. A gradual shift in the stoichiometry may carboxyl end of the NF-H rod domain only disrupts the eventually lead to the progressive formation of neurofibrillary tangles in motor neuron NF-M containing filamentous network. disease or in other neurological disorders such as Parkinson's or Alzheimers disease. Supported by NIH and the Les Turner ALS Foundation.

1009 1010 NORMAL AXONAL DIAMTER REQURES A BALANCED ORGANIZATION AND PHOSPHORYLATION OF AXONAL COMPOSMIION OF NEUROFILAMAENTSUBUNITS NF-L, NF-M AND NEUROFILAMENTS: MODULATION BY MYELINATION. ((S.-T. NP-H ((Z.-S. Xu', P.C. Wong', J. Marszalekt, T. Crawford2 and D.W. Hsieh, G.J. Kidd, T.O. Crawford, Z. Xu, B.D. Trapp, D.W. Cleveland, Cleveland',)) Departments of 'Biological Chemistry, 2Neurology and J.W. Griffin)) Departments of Neuroscience, Biological Chemistry, and 3Neuroscence, The Johns Hopkins Uriversity School of Medicine, 725 Neurology, The Johns Hopkins University School of Medicine, Baltimore, N. Wolfe St., Baltimore, MD 21205. MD, 21287

Neurofilaments are neuronal intermediate filaments comprised ofthree Previous studies have suggested that demyelinated axons are smaller than polypeptide subunits NF-L, NF-M and NF-H. Incre evidence has the normal ones, and neurofilaments in demyelinated axons are more demonstrated that neurofilaments are required for the establishment of closely packed and less phosphorylated than in the myelinated axons. To normal axonal diameter. To test the role of each individual subunit, we examine the effect of myelination on neurofilament organization have constructed transgenic mouse lines that express higher levels of (neurofilament number and spacing) and phosphorylation in the normal NF-L, NF-M or NF-H. By mating mice from different lines, we have peripheral nervous system, we compared the nonmyelinated axonal stem also produced mice that express approximately twice the normal process of the large sensory neurons in the dorsal root ganglion with their amount of both NF-L and NP-M or NP-L and NF-H. Measurement of myelinated intemodes. Axonal caliber and neurofilament numbers were axonal diameters in ventral roots shows that increasing NF-L alone substantially greater in the myelinated internodes than in the stem process inhibits radial growth modestly, but increasing NF-M alone produces or nodes of Ranvier. Neurofilament were closely packed in the stem much smaller axonal diameters. When levels of both subunits are process, similar to those in the nodes; the mean filament-filament spacings increased, normal axonal diameter is restored. In conjunction with were 25-50% greater in the myelinated internodes than in the stem process similar observations in mice that accumulate excessive levels of NF-L or nodes of Ranvier. In the myelinated internodes, neurofilaments had and NP-H, thse data demonstrate that 1) the three neurofilament greater immunoreactivity for phosphorylated epitopes than those in the subunits are not functionally interchangeable and 2) radial axonal stem process. ThIse findings indicate that interactions with Schwann cells growth requires a balanced composition of the three neurofilament modulate neurofilament phosphorylation within the ensheathed axonal subunits. segments, and that increased phosphorylation within myelinated internodes leads to greater interfilament spacing, greater number of neuroftlaments, and thereby greater axonal caliber. 174a Neurofilament Proteins (1011-1016). Monday

1011 1012 INVOLVEMENT OF NEUROFILAENTS IN MOTOR NEURON VARIANT ALLELE OF THE NEUROFILAMENT HEAVY GENE DISEASE: A POINT MUTATION IN A NEUROFILAMENT WHCH PREDISPOSE TO AMYOTROPHIC LATERAL SCLEROSIS. SUBUNIT CAUSES MASSIVE, SELECTIVE MOTOR NEURON (DA Figlewiczl, A. Krizsl, V. Meinnger2, V. Kicinl, GA. Rouleaul and DEATH. ((M. K. Lee, J. Marszalek, and D. W. Cleveland +)) J.-P. Julienl). lCentre for Research in Neuroscience, McGill University, Departments of Biological Chemistry and Neuroscience+, The Johns Montreal General Hospital Research Institute, Montreal, Canada and Hopkins University School of Medicine, Baltimore, MD 21205. 2Centre SLA, H6tel Dieu de Paris, Paris, France. Overexpression of the human neurofilament heavy (NF-H) gene in Two lines of evidence have combined to suggest a direct role of transgenic mice was found to result in a progressive neuronopathy that neurofilaments (NF) in motor neuron diseases such as amyotrophic resembles amyotrophic lateral sclerosis (C6te et al., (1993), Cell 73:3546). lateral sclerosis (ALS). First, abnormal accumulations of NF are an The NF-H protein includes a unique phosphorylation domain of muldple early hallmark of the pathogenic process. Second, forced KSP repeats located in the side-arms which appear to modulate the spacing overexpression of neurofilament subunits in transgenic mice models between neurofilaments. The published sequence of the human NF-H leads to motor neuron dysfunction, albeit without the widespread contains 43 KSP repeats. However, we identified an NF-H allelic variant neuronal death typical of late stage human disease. Using transgenic encoding an additional KSP phosphorylation motif. We have examined the distnrbution of the 43 and 44 KSP NF-H alleles in DNA samples from 148 mice, we now show that accumulation of a modest level (i50%o of control individuals and from 273 non-related individuals with sporadic AIS. amount an mutant the normal of NF-L) of assembly-disrupting point Our data indicate a significant difference in allelic distnbution between the in neurofilament subunit NF-L causes massive degeneration of spinal two groups. In addition, in three ALS patients, mutations were identified in motor neuron cell bodies and axons. In the few surviving motor the phosphorylation domain of NF-H. One ALS patient has a mutant NF-H neurons, perkaryal and axonal accumulations of NFs are prominent. allele with a 102 bp deletion including 5 KSP motifs while two other The neuronal pathology is accompanied by severe denervation- unrelated ALS patients have a mutant NF-H allele with a 3 bp deletion induced atrophy of the skeletal muscles. Like the human diseases, encoding a Lys residue. It is plausible that these mutations alter the cross- sensory neurons remain largely unaffected, despite accumulation of linldng properties of NF-H resulting in an impairment of neurofflament the mutant NF-L subunit. These efforts prove that NF mutations can transport, a mechanism pertinent to the ALS symptoms provoked by an up- in cause selective motor neuron death and that abnormalites in NF may regulation of NF-H cross-linkers transgenic mice. underlie human motor neuron disease.

1013 1014 FATE OF NEUROFILAMENT-M AND -H IN CULTURED NEURONS FROM MUTANT MULTIPLE INTERACTIONS OF ALUMINUM WITH NEUROFILAMENT SUBUNITS: REGULATION BY JAPANESE QUAIL LACKING NEUROFILAMENT-L. ((H. Yamasaki, M.F. PHOSPHATE-DEPENDENT INTERACllONS BETWEEN C-TERMINAL EXTENSIONS OF THE HIGH Ventura and G.S. Bennett)) Department of Anatomy and Cell AND MIDDLE MOLECULAR WEIGHT SUBUNITS ((1B Shea, ML Beermann, F-S Wang )) McLean Biology, University of Florida, Gainesville, FL 32610. Hospital, Harvard Medical School,Belmont MA

Neural tissue from the Quv (quiver) mutant line of Japanese Aluminum exposure induces thedevebpment of periiaryal neurofilament (NP) accumulations. Insight quail exhibits no neurofilaments (NF) in electron micrographs. into the mechanism bywhich this occurs has been provided by studies on interaction of aluminum No NF-L is detectable by immunohistochemistry or immunoblotting with isolated cytoskeletons, purified NFs and dissociated NF subunits. Previous studies have and NF-M and NF-H are severely reduced (Yamasaki et al, Lab demonstrated thataluminum salts markedly decreasetheelectrophoretic migration ofNFs on SDSgeIs Invest 66: 734). The primary defect is a mutation in the NF-L in a time- and concentration-dependent manner, and have suggested that aluminum-induced gene (Ohara et al, J Cell Biol 121:387). In order to investi- stabilizatisn of interactions between protrudingC-terminal eztnsionsofhigh (NF-H) and middle (NF- gate how this defect in NF-L affects the expression of NF-M and M) subunits (either H-H, M-M or H-M) mediate one facet of aluminum interation with NFs. In the NF-H, we have carried out metabolic and immunological analyses present study, wefurthere,nmined the nature of such interctions. of NP-H of cultured sensory (DRG) and spinal cord (SC) neurons from Quv Co-incubation and NF-M and normal embryos. The morphological development of both types but not low (NF-L) molecular weight neurofilament subunits prevents this AIC13-induced altention in of neurons was indistinguishable in Quv and normal. As in electrophoretic migration, suggesting that specific interactions between NF-H and NF-M other than adult tissue, immunoblotting revealed a lack of NF-L in both filament formation influenced their interaction with AIC13. Co-incubation of the 160kDa NF-H a- types of Quv cultures. The level of NF-M and NF-H was also chymotryptic cleavage product (i.e., theC-terminal sidearm) with native NP-M prevented alteration in greatly dimished in Quv SC but, surprisingly, only slightly subunit electrophoretic migration byAK4. Bycontrast, intact, dephosphorylated NF-Hsubunitswere decreased in Quv DRG. 35S-pulse/chase experiments revealed that unable to preventAlC13-induced alteration of native NF-M electrophoretic migration. Taken together, NF-M was synthesized at normal levels in Quv SC and DRG. In these findings suggest that phosphate-dependent interactionsbetween the sidearm extensions of NF- Quv SC most of the pulse labeled NF-M was degraded within 24 Hand NF-M diminish the abilityofAlCA3to associate witheither subunit in a manner that altas their hours, and very little was incorporated into the cytoskeleton. electrophoretic migration. This interaction of NP-H and NF-M sidearms is SDS-sensitive, while AIC13- Although there was a delay in the incorporation of labeled NF-M inducedalteration in elecmphoretic migration of individualsubunits is into the insoluble cytoskeleton of Quv DRG, the rate of NF-M SS-resistant. Addition ofSDS to mixtures of NP-H and NP-M subunits disrupted the protective effect, and promoted degradation was normal. Issunohistochemistry revealed only AlCa3Yinduced subtle differences between Quv and normal DRG cultures, while alterations in subunit electrophoretic migration. These findings support and extend current neurites in Quv SC cultures had markedly dimished NF-M. Many hypothesis that theabily ofaluminum to interact with neurofilamentsubunits isafunctionofsubunit Quv SC cell bodies contained hypophosphorylated NF-M, but more phosphorylation, assembly, andextentofneurofilament-neurofllamentcross-lnking diffusely distributed than in control SC cell bodies.

1015 1016 SITE-SPECIFIC RAPID TURNOVER OF AMINO TERMINAL PHOSPHATE IMPAIRMENT OF AXONAL TRANSPORT IN TRANSGENIC MICE GROUPS ON NF-M SUBUNrr OF NEUROFILAMENTS DURING AXONAL EXPRESSING THE HUMAN NF-H GENE. ((F. C8t6 and J.-P. TRANSPORT. IDENTIFICATION OF PUTATIVE PHOSPHORYLATION SITES. Julien)) Centre for Research in Neuroscience, Montreal ((R.K. Sihag and R.A. Nixon)) McLean Hosp., Havar Med. Sch., Belmont, MA General Hospital, McGill University, Montreal, Canada.

The nurofilament proteins are extensively phosphorylated before they enter the We have produced four lines of tranagenic mice bearing a 34 kb fragment proximal axons. To undertand the function of theae eady phosphorylation events, we that includes the human are studying the dynamics of phosphate groups omneurofilment proteins in iws. neurofilament heavy chain gene (Cell 73:35,1993). Electron microscopy revealed Retinl gangon cel were selcively plse-labeed in viv by injecting mice abnormal neurofilamentous intravitreally swellings in spinal cord motor neurons and with "P-orthophospae. Radiohbeled nuofilament proteins transported in dorsal root ganglion (DRG) cells of the NF-H into optic axons at intervals between 10 ad 90 h postinjection were isolated and transgenics. To determine if the swellings result in a defect in seprated on 5-15% linear gradient SDS-PAGE. NF-M digests were analyzed by two- axonal transport, MuS labeled methionine was injected dimensionl phosphopeptide NF-M was initially in at mapping. phosphorylated uwo into L5 DRGs of normal and tranagenic mice. After 15 or more polypeptide stes.The turnover of'P-phospbate groups from several NH2- various time intervals, mice were sacrificed and terminus siteswas much more rapid than that at the C-terminus sites. By day 4,32P- sciatic nerves and DRGs were removed. Sciatic nerves were removed almostcompletely from two and M2) pbosphae groups major (MI were cut into consecutive 5 m segments and the sites. We have shown that aIro Ml and M2 were previously is sitas phosphorylated protein composition of each segment was resolved using by protein kinase A and protein kinae C but very poorly or not at all by the SDS-PAGE and fluorography. The results showed that the endogenous indePedt kinase(s) which phosphorylated most NF-M sites identified movement of newly synthesized NF proteins along the by In vivo labeling (Sihag and Nixon, JBC 265:4166-4171). To identify the sites of axon occured at the expected rate of lmm/day in the phosphoryltion on phosphopeptide M, andMK, the NF-M subunitwas phosphorylated normal mice. In contrast, in transgenic mice the by protein kinase A. Two '2-P-abeled chymotryptic peptides were isolated by RPLC overall rate of NF transport was significantly on a C,6 reverse-phase column. The P-peptides were sequenced by automated Edman reduced. Surprisingly, the triplet NP proteins were degradaion or counted dircdy aftereach Edman cycle without PTH-analysis. Four not transported in their normal ratio. Approximately potein kdie A phosphoryltion stes at Ser-25, Ser-33, Ser-41 and Ser-49 were half the normal level of NF-L protein was transported identifed. The rapidturoover of specific phosphorylaion sites soon after entering the in transgenic axons and at a reduced rate of In very low proximal axons suggeost a role for these phosat groups in ealy events of 0.6nm/day. addition, levels of radio- neurofilamet assembly and/orthe entry of neurofiaments into axons. NIWNSF labeled NF-M proteins were detected in axons. Proteins 175a Monday.- .1- Neurofilament (1017-1022)I

1017 1018 GLYCOSYLATION OF MAMMALIAN NEUROFILAMENTS PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ((D.L.-Y. Dong§, Z.-S. Xu§, M.R. Chevriert, R.J. Cottert, D.W. ANTIBODIES TO NEUROFILAMENT-ASSOCIATED PROTEINS. Cleveland§ and G.W. Hart§)) Departments of §Biological Chemistry and ((R.Starr1t2,3, J.Xiao2'3, C.E.Hicks23 and M.J.Monteiro"' 3.)) 'Department of tPharmacology and Molecular Sciences, The Johns Hopkins University Human Genetics, 2Medical Biotechnology Center and 3Department of School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205. Neurology, University of Maryland at Baltimore, Maryland 21201.

Neurofilaments (NFs) are neuronal intermediate filaments that play an Neurofilaments (NF) are the major cytoskeletal component of neuronal cells. important role in the growth and maintenance of large myelinated While the structural properties of NF subunits are well documented, the axons. Mammalian NFs are composed of three subunits, NF-L, NF-M functions of these abundant elements remains enigmatic. In an effort to better and NF-H, all of which are phosphorylated. Here, we demonstrate by understand NF function, we have generated a panel of monoclonal antibodies several criteria that all three NF polypeptides are also modified by an (MAbs) specific for proteins that associate with NF. NF-associated proteins abundant type of intracellular protein glycosylation in which single N- were obtained by passing a mouse brain extract over an affinity column to acetylglucosamine monosaccharides are 0-glycosidically (O-GlcNAc) which NF-H protein was covalently bound. The NF-associated proteins eluted linked to or residues. In purified NFs, the O-GlcNAc from the column were used to immunize mice for the production of MAbs. modifications occur at a stoichiometry of approximately 0.1, 0.15 and A number of these MAbs reacted by Western blotting with distinct 0.25 mole of GlcNAc per mole NF-L, NF-M, and NP-H, respectively. polypeptides from both mouse and human brain but not with proteins from The predominant sites of O-GlcNAc attachment on NF-L and NF-M other tissues examined. Further, the ligands recognized by these MAbs were have been identified. For NF-L, three glycosylation sites (Thr 21, Ser shown to be associated with NF by immunoprecipitation, using the reversible 27 and one or more Ser between 44-52) are all located in the N-terminal homobifunctional cross-linking agent dithiobis (succinimidyl propionate), head domain. For NF-M, one site (Thr 48) lies within the head, while whereas a control ligand was not associated by this criterion. We used the a second site (Thr 431) is located in the tail just next to the end of the MAbs to determine the distribution of their ligands in mouse brain sections rod. Deletions encompassing these sites have previously been shown by immunocytochemistry. Most of the MAbs were specific for neuronal cells, to have a dominant detrimental effect upon NF assembly, raising the consistent with the restricted expression of NF in these cells. Interestingly, possibility that addition of saccharide moieties to NPs in nerve cell some ligands were only expressed in certain neuronal populations. We are bodies and axons may alter assembly, stability or transport properties. further characterizing the NF-associated proteins recognized by these MAbs in order to gain insight into their role in neuronal function.

1019 1020 CHARACTERIZATION OF A NOVEL NEUROFILAMENT- IDENTIFICATION OF A NEUROFILAMENT-SPECIFIC CDC2-RELATED MCB KINASE REGULATORY PROTEIN EXCLUSIVELY EXPRESSED IN ASSOCIATED KINASE (NAKl 15). ((. Xiao and M.J. Monteiro)) NEURONS Graduate Program, Medical Biotechnology Center and Department of ((K.T. Shetty, S. Beushausen, W.T. Link, H. Jaffe, C.M. Fores, S. Wray and H.C. Neurology, University of Maryland at Baltimore, Maryland 21201. Pant.)) LNC and LN, NINDS, National Institutes of Health, Behesdda MD 20892. Neurofilament proteins, NF-H and NF-M, contain casboxy tail regions rich in KSP Neurofilaments (NP) comprised of three principal subunits, NF-H, NF-M sequences, characterized by two pentapeptide motifs, KSPXK and KSPXX. and NF-L, are the major intermediate filaments expressed in neuronal cells. Phosphorylation of these sites is presumed to effect intertactions with other Although NF are one of the most highly phosphorylated proteins expressed cytoskeletal components. We have identified a cdc2-like kinase activity from rat in brain, little is known of the physiological function of this modification. spinal cord that specifically phosphorylates KSPXK neurofilament motifs. The enzyme copurifies with and has activity-dependence upon a protein of MW 62 kD, In order to understand the role of NF phosphorylation and the kinases that (p62), as sessed by SDS-PAGE. Separation of p62 from the kinase results in a phosphorylate NF in vio, we used bacterially exprssed NF-H as a affinity significant diminution of KSPXK-directed kinase activity that is restored by ligand to isolate proteins from mouse brain that interact with NF. Using reconstitution with purified p62. Oligonucleotide primers were used to synthesize DNA from rat brain mRNA using RT-PCR based on amino acid sequence this approach a number of proteins that specifically bound to NF-H were information obtained from HPLC-purified p62 . Full length coding sequence of isolated. These proteins were enriched for kinases able to efficiently cDNA clones predicted a protein of MW 67.5 kD consistent with the observed phosphorylate NF proteins in vitro. We characterized these kinases further migration of antigen in whole brain extracts foliowing Western blot analysis using a the on and p62 peptide-specific antibody. Protein database searches reveal that p62 is a novel by separating proteins denaturing polyacrylamide gels protein unrelated to cyclins. In-situ hybridizadon studies with mouse embryos and reconstituting kinase activity in situ. By this assay we identified at least 15 adults de d that p62 transcriptexpsion begins ealy in develpment and is individual kinases that bound to NF-H. Of these kinases a 115 kDa resuiclod to the nervous system. Moreover. immunocytochemical double labeling of polypeptide was found to have a particular preference for NF proteins as the trigeminal ganglia following in-situ hybridization with antisera to low MW neurfament protein or penipherin demonstrated that p62 expression is remstricted to a substrate. The 115 kDa knase, which we have termed NAK1 15, differs neurons. These data suggest evidence for a regulator, other than a cyclin, of a cdc2- in a number of properties from the other kinases that have been shown to like kinase activity that is exclusively expressed in neurons that may play a role in phosphorylate NF in vitro. Further, we have confirmed that NAK115 is addition or unrelated to maintaminence of the cel cycle. indeed associated with NF in viw since the kinase co-immunoprecipitates with NF. Finally a kinase of similar size and properties has been found in human brain lysates indicating that this kinase is conserved across species.

1021 1022 USE OF RECOMBINANT FUSION PROTEINS TOWARDS SEQUENCE, STRUCTURE AND TARGETED MUTAGENESIS OF THE DETERMINING SITES PHOSPHORYLQTED BY A NJUROFILAMENT- MOUSE NEURONAL INTERMEDIATE FILAMENT a- ASSOCIATED KINASE. ((B.A. Hollander, G.S. pennett, and G. Shaw)) GENE. Chien and R.K.H. of Dept. of Anatomy and Cell Biology and Dept. of Neuroscience, ((C.L Liem)) Department Pathology, University of Florida College of Medicine, Gainesville, FL, 32610. Columbia University, New York, NY 10032. The carboxy-terminal tail of chicken NF-M, the middle molecular mass We have determined the complete nucleotide sequence of member of the neurofilament triplet proteins, contains 22 putative phosphorylation motifs: a single KSD, 4 KSP repeats, and 17 repeats of the mouse gene encoding a-internexin. The mouse the consensus KXX(S/T)P. We are using recombinant fusion proteins to a-internexin gene contains three exons and two introns, and characterize an NF-associated kinase (NFAK) partially purified from both can be classified as a IV intermediate filament. The chicken and mammalian sources. NFAK phosphorylates, in vitro, serine nucleotide of the mouse a-intemexin shows but not threonine in a fusion protein containing the KSD and 4 KSP sequence gene repeats (CM:381-805) and one that contains only the KSD sequence high homology to that of rat. Comparison of the deduced (CM:381-588), but not one that contains only the 17 KXX(S/T)P sites amino acid sequences between mouse and rat genes reveals (CM:594-858). NFAK-mediated phosphorylation of NF-M, as well as the that the head and rod domains are highly conserved. fusion proteins, is strongly inhibited by CKI-7, a specific inhibitor of casein the tail domains show differences kinase 1. Two dimensional phosphopeptide maps of NF-M, However, significant dephosphorylated NF-M, CM:381-805, and CM:381-588, all which may make it possible to raise specific antibodies to phosphorylated in vitro with NFAK, yield the identical subset of the mouse a-intemexin protein. phosphopeptides that are obtained from NF-M phosphorylated in vivo. The mouse a-internexin genomic clone has been used for motifs as sites These resuits exclude KSP and KXX(S/T)P phosphorylated a by NFAK, leaving the KSD and/or another serine(s) not conforming to construction of replacement vector to target the known phosphorylation motifs as the phosphorylation site(s). The data a-internexin locus within embryonic stem (ES) cells by also provide experimental evidence that multiple kinases are responsible homologous recombinatlon. The targeted ES cells have been for NF-M tail phosphorylation, at least one of which is related to casein selected by PCR and Southem blot assays. The generatlon of kinase 1. transgenic mice is now in progress. 176a Neurofilament Proteins (1023). Monday

1023 THE EFFECTS OF THE REMOVAL OF THE HEAD AND TAIL DOMAINS OF a-INTERNE)GN ON TYPE IV NEURONAL IF NETWORK FORMATION. ((G.Y. Ching and R.K.H. Uem)) Department of Pathology, Columbia University, New York, NY 10032. Uke the neurofilament triplet proteins (NFTPs) NF-L, NF-M, and NF-H, oa-internexin Is a type IV neuronal intermediate filament (IF) protein. We previously showed that a-internexin was capable of forming a filament network on its own and coassembling with each of the NFTP subunits to form filamentous networks. In contrast, none of the NFTP subunits was able to self-assemble Into a filament network. However, coassembly of NFH with NF-M or NF-H, but not that of NF-M with NF-H, resulted In formation of filament networks (ChNng and Lem, J. Cell Biol. in press). We present here the assembly studies of the amino- and carboxyl-terminal deletion mutants of a-intemexin lacking the head or tall domains In a non-neuronal cell line in the absence of any pre-existing cytoplasmic IFs. Our results show that the headless a-intemexin mutant lost the competency to self-assemble into a filament network, yielding a punctate Immunofluorescence pattem. The tailless a-intemexin mutant formed aberrant aggregates which appeared to consist of filament bundles. Interestingly, NF-M was the only subunit among the NFTPs that could form filament networks with both mutants. Cell Receptors and Extracellular Matrix I (1024-1027)

1024 1025 ALTERED EXPRESSION OF CELL ADHESION MOLECULES ON HUMAN LYMPHOCYTES BIND TO NEURAL CELL ADHESION MOLECULES CD4+ AND CD8+ LYMPHOCYTES IN A 3D COLLAGEN MATRIX. ON VIRUS-INFECTED NEURONS ((Bryan M. Gebhardt)) LSU Eye ((P. Friedl', P. B. Noble2, B. Niggemann3, and K. S. Zanker')) ' Institut for Center, LSU Medical Center School of Medicine, New Orleans, LA 70112 Immunologie, UniversitAt Witten/Herdecke, Germany, 2 McGill University, 3 Montreal, PQ, Canada, Evangelisches Waldkrankenhaus, Bedin, Germany. Lymphocytes migrate into and localize around herpes simplex virus type The modulatory role of the extracellular matrix (ECM) on various cell adhesion 1 (HSV-l)-infected neurons in the central and peripheral neural tissues of molecules and integrns of human CD4+ and CD8+ lymphocytes was inves- infected mice. Previous studies indicated that HSV-1-infected neurons tigated. Immunomagnetically isolated peripheral CD4+ and CD8+ lymphocytes express increased quantities of neural cell adhesion molecules (NCAMs). were incorporated into 3D hydrated collagen gels prior to polymerization or The present study was undertaken to determine if lymphocyte homing and Incubated in RPMI 1640 medium/5% FCS (collagen-free control). Lymphocyte binding to virus-infected neurons is a function of ligand-receptor (LFA- migration in collagen was observed for 1 to 8 days. Subsequently the cells NCAM) interaction. Neural tissues sections from virus-infected mice were were harvested by collagenase digestfon and washing procedure. ECM receptor expression was analysed by two-colour flow cytometry. incubated in the presence of lymphocytes from the infected animal. It was Lymphocyte motility in collagen peaked after 2 to 5 days in the matrx and found that lymphocytes attached to neurons at a significantly greater declined after day 6 to low levels. Prolongued exposure (4 to 8 days) of frequency (p<0.005) in virus-infected tissue, compared to uninfected lymphocytes to collagen led to a continuous reduction of CD44 and L-selectin tissue. This binding of lymphocytes to neurons in virus-infected tissue could expression to 15 - 25% of the collagen-free controls. For a minor lymphocyte be blocked either by incubation of the lymphocytes in an anti-LFA antibody subpopulaffon CD44 was transiently up-regulated. In addition, both, CD4+ and or by incubation of the tissue sections in anti-NCAM antibody. Studies CD8 lymphocytes showed a signiicant up-regulation of VLA-2 (CDw49b) and conducted in tissue culture with isolated neurons gave similar results, ie, CD29 (100 to 500%) after 4 to 6 days in the collagen gel. VLA-3, -4, -5, and -6, CD27 and CD28 were not significantly altered in comparison to the collagen- lymphocytes bound to virus-infected neurons and could be prevented from free controls. The modulation of VLA-2 and CD29 as well as CD44 and L- doing so by pretreatment in anti-LFA antibody. In vivo studies in which selectin on T cells was enhanced if the PBMC fraction instead of isolated CD4+ virus-infected mice were treated with an anti-NCAM antibody resulted in and CD8+ cells was investigated suggesting an additional modulatory function lowered frequency of lymphocytes in the neural tissue and the presence of of bystanding cells. The collagenase digestion procedure itself did not affect the fewer lymphocytes bound to virus-infected neurons. These studies indicate expression of any of the molecules investigated. that a part of the homing and localization mechanism of lymphocyte In conclusion, exposure of resting perpheral lymphocytes in an in vitro collagen and behavior. recognition of virus-infected neurons is due to the LFA-NCAM interaction matrix may contribute to alterations in lymphocyte phenotype at the cell membrane. (Supported in part by NIH/NEI grant EY08701).

1026 1027 THE PAMYLOID PRECURSOR PROTEIN BINDS TO THE IKVAV EXTRACELLULAR MATRIX (ECM) REGULATES MECHANOSENSITIVITY OF BIOLOGICALLY ACTIVE SITE OF LAMININ AND FUNCTIONS IN MESANGIAL CELLS (MCs). V. Chen, H. Guber, C.E. Palant. Dept. NEURITE OUTGROWTH. ((N.C. Kibbey, M. Jucker, B.S. Med. VAMC and SUNY Health Science Center at Brooklyn, NY. Weeks, R.L. Neve, W.E. Van Nostrand, and H.K. National Institute of Transmission of mechanical forces from the ECH to membrane Kleinman)) Lab. Devel. Biol., receptors, critically influences cellular growth and function. Dental Research, NIH, Bethesda MD 20892; Swiss Fed. To further study cell membrane interactions with the ECM that Inst. Tech., Zurich Switzerland; McLean Hospital, may be required for mechanotransduction, we asked whether Belmont MA 02178; UCQIrvine CA 92717. mechanoactivated ion channels (MACs) were modulated by ECM proteins. Rat MCs in third to eight culture passages were We previously described a 110 Kd binding protein plated in dishes coated with one of the following ECM's: (1) (LBP110) from mouse neonate brain for the IKVAV mouse collagen IV (COL); (2) human fibronectin (FN); (3) mouse containing site of the laminin A chain (Kleinman et laminin (LM); (4) a mixture of 60 LM, 30S COL, 3X Heparan- al., Arch. Biocehe. Diophys. 290:320-325, 1991). Due S04, 1% entactin and 6% vitro- nectin, TGF-B, FGF-f and TPA to similarities in localization of both LBP110 and (Hatrigel) and cultured in serum supplemented media. MCs were the precursor (APP) under both bathed in NaCl Ringer's and electronically monitored negative Uamyloid protein pressures (-P) delivered through cell-attached patch-clamp normal conditions and following injury, we electrodes, used for recording of single ion channels. Pipet investigated if LBP110 was a member of the APP solution contained (in mM): Na-gluc. 135, CsCl 15, HEPES 10, family. Affinity-purified LBP110 was recognized by Ph 7.4. MACs were detected in 3/10 cells In COL, 8/9 cells In 4 different APP antisera raised against different APP FN, 3/4 cells in Matrigel, 8/10 cells in LM. MAC responses in epitopes. Both recombinant and purified APP were COL were significantly delayed requiring higher membrane recognized by LBP110 antiserum. By ligand blot, APP tension levels. MAd kinetics were analyzed in 41 episodes of specifically bound IKVAV-containing peptide. NGF- suction (>2 X 10 events) in 11 cells. Cumulative MAC open treated PC12 cells had elevated LBP110-immunoreactive probabilities in FN 10.84 at -P=24 mmHg) were higher than in similar to that reported for APP. COL (0.19 at -P=42 mmHg, p<0.05) and LM (0.21 at -P=27 mwHg, protein previously p

1028 1029 DIFFERENTIAL USE OF INTEGRINS AND SYNTHETIC PEPTIDES ON IMMIUNOLOCAI ZATION OF CD44 IN TEM VRTEBRATEI MURINE AND HUMAN LAMININ-INDUCED CHEMOTAXIS IN RETINA. ((M.H. Chaitin. H.S. Wortham. M.T. Ankrum and A.M. HT1080 HUMAN FIBROSARCOMA CELLS. ((M.-L. Lin and L. T. Zinkernagel)) Dept. of Anatomy and Cell Biology, Univ. of North Furcht)) Department of Laboratory Medicine and Pathology, University of Texas Health Science Center at Fort Worth, Fort Worth, TX Minnesota, Minneapolis, MN 55455 The transmembrane glycoprotein CD44 is a cell adhesion Previous studies have shown that human cells use Q301 integrin molecule which has recently been localized in a variety of cell preferentially to bind human laminin (H-LMN), whereas they use rx6plI types. It mediates cell attachment to extracellular matrix integrin to bind murine laminin (M-LMN). Different anti-integrin antibodies components and also binds to the actin cytoskeleton within the are used in this study to examine the integrins involved in M- and H-LMN- cell. In this study, we investigated the presence of CD44 in induced chemotaxis in HTl080 human fibrosarcoma cells. The results show mouse and rat retinas to determine if this molecule might be that anti-pl and anti-P4 antibodies inhibit cell migration to both M- and H- important for retinal cell adhesion. With immunoperwxidase LMN (.50%), whereas anti-a6 antibody only inhibits migration to M-LMN techniques, positive labeling for CD44 was found at the level of (-80%), and anti-a3 antibody inhibits migration to H-LMN (-50%). This the outer limiting membrane and in the region just above it. suggests that a3PI and a6cI may have different affinities to M- and H-LMN. To further understand the different affinities of both integrins, it is important However, from these light microscope results it was not clear if was in inner cell to know their binding sites in LMN. As the binding domain for a3p I and a6poI the label the photoreceptor segments, Muller has been located in the carboxyl-terminal globular domain at the end of long processes. or both. In order to answer this question, arm, synthetic peptides from this region are used to examine their effects on cryoultramicrotomy and immunogold labeling were used to laminin-induced chemotaxis. Peptides GD-1, GD-6 and GD-7 inhibit demonstrate that CD44 is specifically localized to the Muller chemotaxis to M-LMN (-80%), whereas they only exhibit 20-40% inhibition cell processes which appose the interphotoreceptor matrix. on H-LMN. Peptide GD-5 enhances cell migration to M-LMN, yet it slightly Westem blotting showed that the anti-CD44 is specific for a inhibits migration to H-LMN. Moreover, peptides GD-3 inhibits chemotaxis protein of 80-90 kilodaltons which is the correct molecular to both M- and H-LMN by -80%. The data suggest that these peptides may weight for CD44H, the most abundant form of CD44. These bind to different integrins or to different domains of the same integrin which results demonstrate the presence of CD" in the Muller cell may then cooperatively regulate the biological function of laminin counter processes of the mouse and rat retina and suggest that CD44 receptor binding. Using these peptides can help us to localize the precise could play a role in mediating the attachment of the neural laminin binding sites for a3P and "6PI integrins, and to understand the mechanism of preferential interaction of human cells to M- or H-LMN retina to components of the interphotoreceptor matrix. (supported by NIH-CA-29995).

1030 1031 HYALURONIC ACID RECEPTOR, GP116, IS A NEW CD44 VARIANT IN OLIGOMANNOSIDES INITIATE CELL SPREADING OF LAMININ-ADHERENT MURINE ENDOTHELIAL CELLS. ((V.B. Lokeshwar, and L.Y.W. Bourguignon)) MELANOMA CELLS. Department of Cell Biology and Anatomy, University of Miami ((S. Chandrasekaran', M. S. Ginigert and M. L Tanzerl))lDept. of BioStructure & Function, Medical Miami, FL 33101. (Spon. by R.W. Rubin). University of Connecticut Health Center, Farmington, CT; tDept. of Oral Medicine, LSU School, Medical Center, New Orleans, LA.

In this study, we have used a variety of biochemical, Murine melanoma cells readily bind and spread on murne lariinin. Laminin glycofoms which immunocytochemical and molecular biology techniques to contain imrnature oligosaccharides seem more effective in restoring cell spreading than do establish that OP116, a hyaluronic acid (HA) receptor may be more mature laminin glycoforms. We have produced and characterized an oligomannoside- important for endothelial cell activation. Structurally, GP116 rich glycoform, derived from murine laminin-synthesizing cells incubated In kifunensine, a potent inhibitor of mannosidase 1. Studies using endoglycosidase H and ledin blotting show (glycoprotein of 116 kDa) shares immunological cross- that this purified laminin glycoform is enriched in high mannose oligosaccharides. When used reactivity with a panel of CD44 antibodies indicating that it as a substratum for cell attachment and spreading, it was as effective as mature is a CD44-like protein. Using a standard pulse-chase protocol, glycosylated Iaminin, which primarily contains complex oligosaccharides and small amounts we have detected a 50 kDa polypeptide precursor (p50) which is of high mannose oligosaccharides. When added in solution to unglycosylated laminin- different from that of standard hemopoietic CD44 (CD44s). The adherent cells, high mannose laminin was more effective in promoting cel spreading than mature glycosylated Reconstitution experiments with showed that cell conversion of p50 to GP116 requires further glycosylation laminin. saccharides spreading is rapidly initiated, by mannan, a branched polymer of mannose, which ls eff ctive (both complex type N-linked and 0-linked). In addition, RT- In the microgram range. Titration of cellular spreading with mannan yields an adsorption PCR, cloning and sequencing data suggest that an additional isotherm profile. Cell spreading approaches a maximum level within an hour after initiation by exon is inserted in GP116. This new exon shares sequence saturating levels of mannan. Of the various polysacchrides examined only mannan was able homology with human CD44 exon 14. Amino acid composition to restore the spreading of cells on unglycosylated laminin. Mannose is an antagonist, preventing mannan from restoring cell spreading, but is not an agonist, High mannose- analysis of purified GP116 have further confirmed the containing glycopeptides ware depleted from a pronase digest of laminin by lectin affinty expression of the additional exon 14. Taken together, these chromatography. The depleted digest was unable to restore cell spreading while a control data indicate that GP116 is a new CD44 variant isoform digest was fully active. Melanoma cells were unable to bind to three different neoglycoprotein (CD44v). The fact that GP116 is involved in HA-mediated surfaces. However, when soluble unglycosylated laminin was present in the medium, the functions (e.g. binding, cytoskeleton () interaction cells adhered to and spread upon mannosylated-BSA. In contrast, they did not adhere to glucosyl-BSA or galactosyl-BSA in the same assay. Of the various cell lines known to and mitogenexis) suggests that the presence of exon 14 in interact with glycosylated laminin only murine melanoma cells showed oligomannoside- GP116 molecule may be critically important for HA-mediated dependent spreading on an unglycosylated laminin substratum. The composite resuits signal transduction and endothelial cell proliferation. indicate that oligomannosides are necessary to initiate murine melanoma cell spreading on b&minin but are not sufficient for cell adhesion.

1032 1033 THE 67-KD SPLICED VARIANT OFB-GALACTOSIDASE ACTS AS A PROTECIVE THROMBIN INCREASES EXTRACELLULAR MATRIX PROTEIN DEPOSITION COMPANION PROTEIN FOR TROPOELASTIN ((A. Hinek and M. Rabinovitch)) IN CULTURED ENDOTHELIAL CELLS BY A MECH*ANISM INDEPENDENT Division of Cardiovacular Research,The Hospital for Sick Children and Department of OF ITS PROTEOLYTIC ((Es Papadimitriou University of CANADA. ACTIVITrY B.R. Unsworth M.E. Pathology, Tointo, MaragoudakisO, and P.I. Lelkes )) Marquette University, Milwaukee, WI, We have previously shown that the 67-kD cell surface elastin binding protein (EBP) OUniversity of Patras, Medical School, Greece, University of Wisconsin, Medical responsible for cell adhesion to elastin and for mediating the process of elastin fiber School, Milwaukee, WI. assembly issimilar if not identical to the 67-kD catalytically inactive alternatively spliced form of -galactosidase (S-GAL). In thisstudy we have establishedthat an antibody (anti- Thrombin is a serine protease that plays a primary role in hemostasis S-GAL) which recognizes the elstin binding domain of spliced variant of human through the modulation of clot In addition, thrombin can act galactosidase co-immunoprecipitates this 67-kD protein in a complex with 70-kD formation. directly on a variety of tropoelastin from sheep aorta smooth muscle cell extracts. Moreover, S-GAL and cells, including endothelial cells. Cellular responses to thrombin require activation trpoelastin co-loaLize intacellularly, on the cel surface and extracellularly over the of thrombin receptors and may also involve thrombin's proteolytic activity. In the elastic fibers, as assessed by immunofluorescence and immunoelectron microscopy, present study, we examined the effect of thrombin on the deposited extracellular suggesingdat hes two proteins assoclae with each other along thescretory pathways. matrix (ECM) proteins, fibronectin, laminin, collagen-IV and collagen-I in cultures The of this was further established by showing that functional significance association of rat adrenal medulla endothelial cells. dissociation of the EBP-trodastin complex caunad by treaument of SMC lysates with Tbrombin, after 5-minute stimulation of the galactosugars acceerates degradation of free tropoelastin by endogenous serine cells, increased up to three fold the deposited amounts of all the above molecules into proein e(s). Since the GSPAQDEASPLsequence which represents theelastin binding the ECM in a dose-dependent way, in concentrations between 0.03 and 0.25U/ml; at motif of S-GAL also displays homology with N-teminal sequences of serine elastses we higher thrombin concentrations, this effect was gradually reversed. Tne amounts of speculated,that thse protes maydsare a common ligand-binding motif and compete for the individual ECM proteins released into the culture medium were decreased binding of tropoelastin. This was confirmed in an in vitro assay, when preincubation of proportionally to the amounts deposited. The effect of theholo protein was fully [3H]-elastin with purified sheep aort EBP or with GSPAQDEASPL synthetic peptide reflecting the elastin binding motif of S-GAL protected it from degrdation by bovine mimicked by a peptide agonist of the thrombin receptor, which shows that the pancretic elastae (PPE) or human leukocute elamse (HLE). Moreover, blocking of stimulatory effect of thrombin on ECM deposition was mediated by binding to its VGVAPG sequenes on elastin which bind S-GALUEBP orblocking N-teminal domains of receptor. It also did not require the catalytic activity of thrombin, since it was HLE and PPE homolgu to lstin binding doman of S-GAL with antibodies raised to mimicked by a proteolytically inactive enzyme, and was not dependent on de novo the respctive synthetic pepides have prevented binding of elastases to[3H]-elastin and protein or mRNA synthesis. Finally, pharmacological studies suggest that the precluded its degrdto. Thee strongly msuggeat th at the 67-kD protein may thrombin-induced increase of the amounts of the ECM proteins deposited onto the serve as a compnio ptin which protects trpoelatm from premaur degadation and matrix of the cells, involves activation of the adenylate cyclase ases its asmbly an insole componet ofeLic fiber pathway of intracellular signal transduction. 178a Cell Receptors and Extracellular Matrix I (1034-1039). Monday

1034 1035 EXPRESSION OF THROMBOSPONDIN-1 INA TRANSFORMED INTRACELLULAR EXPRESSION OF CD36 IN CYTOKINE-TREATED ENDOTHELIAL CEL LINE RESTORES A NORMAL PHENOTYPE HUMAN MONOCYTES. ((L.M. Yesner, H.Y. Huh, and R.L. Silverstein)) ((N. Sheibani*, A. RayChaudhury+, PA. D'Amore+, and WA. Frazier*)) Department of Medicine, Cornell Medical College, New York, NY 10021. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110 and +Department of CD36 (glycoprotein IV) is an 88 kD integral membrane protein function- Pathology, Harvard Medical School, Boston, MA 02115. ing as an adhesive receptor for thrombospondin, collagen, and oxidized low-density lipoprotein on platelets, monocytes, macrophages, and Thrombospondin-1 (TS1) inhibits angiogenesis in vivo and blocks microvascular endothelium. Because of the potential importance of proliferation and migration of endothelial cells (EC) in vitro (Tolsma et al., J Cell Biol. 122:497, 1993). We have observed that expression of TS1 is CD36 in mediating atherogenic and inflammatory processes, we studied down regulated in polyoma middle T-transformed endothelial (bEND) cells, its mRNA and protein expression in cytokine-treated human peripheral compared to normal EC. However, bEND cells adhere to TSI and their blood monocytes. RNase protection assays showed 7-10 fold increases growth is inhibited by exogenously added TS1. In an attempt to better in CD36 mRNA levels with IL-4, IL-6, M-CSF, and PMA treatment, urerstandthe roleof TSI in the control of growth and differentiation of whereas 3-5 fold decreases in CD36 mRNA levels were seen in LPS and endothelial cells, we have transfected the bEND cells with a TS1 expression dexamethosone treated monocytes. In contrast, no apparent differences plasmid (pcDNATSl) containing the CMV promoter. Co-transfection with in cell surface CD36 antigen levels were detected in subsequent a plasmid encoding hygromycin resistance allowed selection of the already immunofluorescence flow cytometric analysis. In order to localize CD36 G418-resistant bEND cells. Northern analysis of hygromycin resistant protein, cytokine-treated human monocytes were permeabilized and transfectants indicated abundant TSl mRNA. The TSl-transfected cells stained through indirect cytoimmunofluorescence. A dramatic increase exhibited a morphology similar to that of nonnal EC, clearly different from in the intracellular fluorescence was observed with M-CSF and PMA, the bEND cells or bEND cells with the vector either parcntal transfected while LPS showed markedly diminished intracellular intensity. Western alone, both of which displayed a spindle-shaped, fibroblastic appearance. blot analysis on human monocyte lysates using Furthenmore, TSl-transfectants grew more slowly than controls. Expression monoclonal anti-CD36 of TS1 protein was confrmed by ELISA of the conditioned media, Western antibodies confirmed these observations. Intracellular accumulation of blotting of cells and immunofluorescent staining with an anti-TSl antibody. CD36, a cell surface adhesion receptor, suggests a complex, multi-step The redistribution pattern of the adhesion molecule PECAM (CD31), a mechanism of CD36 synthesis and translocation to the plasma mem- marker of EC, was altered in TS1 transfectants. These data suggest a pivotal brane. Elucidating the mechanism of intracellular CD36 transport will role for TSl in the regulation of EC phenotype. (Supported by HL14147*, yield insight into the specific role of CD36 in monocyte function and The Monsanto Co.* and CA45548+). vascular pathogenesis.

1036 1037 CHARACTERIZATION OF 70-KD AMINO-TERMINAL FIBRONECTIN C. ALBICANS YEAST AND GERM TUBE EXPRESS A FIBRONECTIN RECEPTOR FRAGMENT BINDING RECEPTORS ON IN VITRO DIFFERENTIATED ANTIGENICALLY RELATED TO a501 INTEGRIN (-G. Santoni, A. Gismondi, A. Punturieri, L. Jwhong M. Piccoli, OJ. Djeu A. Santoni and L. Frati) *General HUMAN MONOCYTES ((W.G. Chung and J.E. Kaplan)) Department of Pathobgy Chair, Universky of Camenno, Dept of Experinmertal Medicine, University Physiology and Cell Biology, Albany Medical College, Albany, NY 12208 of Rome, Italy, °Moffitt Cancer Certer, Univ South Fbrida, Tampa, Fl, USA

Previous studies in our laboratory have characterized an amino-terminal Cell adhesion molecules by regulating host-microrganism interaction play a major fibronectin binding protein on rat peritoneal macrophages and human U937 role In the pathogenesis of rinectious diseases. present was to cells. In this study we describe a similar binding protein on freshly isolated The study undertaken investigate the expression of the FN receptor prototype co5I hntegrin on C.abicans and its involvemrent In the and cultured human peripheral blood monocytes In vitro. Competitive adhesion to FN. We also analyzed whether changes In the expression and function of this binding studies of the 70-kD amino-terminal fibronectin fragment to the 18 receptor could occur durng yeast-mycelial transtion, a process clsely related with hour cultured monocytes revealed a single class of binding site with KD= increased invasiveness and pathogenicity. 1.31 x 10- M and approximately 9 x 103 binding sites/cell. Binding was FACS analysis using a panel of Mabs directed against the humnan a5 (SAM-1, inhibited by poiyclonal antibody to the previously reported 67-kD recep- P1D6, Mabl6) or P1 (AlA5, 4B4) integrin subunits, or two different antisera arlsed tor(JBC 267:3968). Binding studies have further shown that the specific against the affinity purified FN receptor (FNr) from human placenta, showed both anti-FNr antisera and the anti-aS SAM-1 MAb but not with PID6 and Mab16 binding of the 70-kD fibronectin fragment increases with maturation of the positively stained C. albians yeast; anti-P1MAbs were only marginally reactive. monocytes. When compared with freshly isolated monocytes, two day Expression of a5 and pl subunits was increased (2-4 fold) upon germ tube cultured monocytes show a 5 fold increase in the specific binding of 70-kD transition. 30% of 3H glucose-labeled Candida yeast and germ tube phases fibronectin fragment and three day cuitured monocytes show an increase in specifically adhered to FN and its 12OKd fragment in a time and dose-dependent excess of 15 fold. These increases were inhibitable by preincubating the manner and this adhesion was increased by divalert cations. C.albicans yeast and monocytes with 8-Br-cAMP for 30 minutes at 3rC. One mM concentration germtube adhesion to FN was markedly inhibited by GRGDSP, but not GRGESP The of an of 8-Br-cAMP decreased the binding of 70-kD fibronectin fragment on both peptide. involvement aSp1 integrin-lTke molecule in the adhesion of Candida yeast and germ tube phases to FN was further indicated by the ability of two day and three day cultured monocytes by more than 50% when anti-a5 SAM-1 Mab, or anti-FNr antisera to strongly Inhhbit binding to FN. Overall compared to the controls. These studies demonstrate that monocytes these resuits Indicate that C.albicans yeast and germ tube phases express a express receptor to the amino terminal fragment of fibronectin. The receptor functional a5S1 integrin-like receptor which mediate their adhesion to FN. expression is Increased during differentiation and decreased by cAMP. Upregulation of this receptor during germ tube transition is not assoiated with (NIH P01-GM-40761, T32-HL-07194) increased adhesion to FN. a5pl integrin-lke receptor expression on C.albicans could be relevant for fungus-host interaction and in the dissemination process of Candida infection.

1038 1039 CHARACTERIZATION OF AVIAN FIBRONECTIN RECEPTOR CHARACTERIZATION OF A PEPTlDE HYDROPATHICALLY COMPLEMENTARY SUBUNITSa,,. ANDas,,. ((R. Rajaraman)) Departmentof Medicine TO THE PLATELET RECOGNMON SEQUENCE y402-411 OF HUMAN and Department of Microbiology & Immunology, Faculty of FIBRINOGEN(FG). ((D.B. Taylor1, X Wang2, J.M. Derrick2, and T.K. Medicine, Dalhousie University, Halifax, N.S.. Canada, B3H 4H7. Gartner2)) Department of Biology, Illinois Benedictine College, Usle, IL 605321, Department of Biology, Memphis State University, Memphis, TN Avian fibronectin receptor (FNR) from chicken embryo fibroblasts, 381522. adult and embryonic chicken gizzard, chicken embryo, and chicken embryonic heart were isolated by ligand-affinity chromatography. FG binding to platelets is mediated primarily, if not exclusively, by B, The putativeaL, and subunits of avian FNR had a non-reduced M, residues 400-411 of the FG y chain. A peptide corresponding to FG y4O2- of 165 kD and 110 kD; these co-migrated with humana, and B, 411 can inhibit platelet aggregation and the adhesion of platelets to FG by respectively. The 165 kDaL, subunit yielded two polypeptidesof 145 inNbiting the binding of FG to the integrin,allb/p3. We demonstrated that kD and 30 kD upon reduction; the B, subunit was 130 kD upon the peptide, DPPRFVRPLQ, predicted by the anticomplementarity reduction. In immunoprecipitation studies twoa subunits, i.e.,cx,a hypothesis to bind to FG y4O2-41 1, inhibits platelet aggregation and the (155 kD) anda, (165 kD) could be recognized; the 165 kdaS5b adhesion of stimulated platelets to FG. Anti-DPPRFVRPLQ antibodies also subunit yielded two polypeptides of 145 kD and 30 kD upon inhibit platelet aggregation and adhesion (J. Cell Biol. 109: 1 7a, 1989). reduction and is probably identical to the affinity purified 165 kD The present studies reveal that DPPRFVRPLQ may be a FG specific peptide. a, subunit. This 165 kD oL, subunit resembles the humanoa, in that At peptide concentrations exceeding theIC50 ofinhibition of platelet it is FN-and RGD-specific, and is composed of two disulfide-linked adhesion to FG, the peptidedidnot inhibit the adhesion of stimulated polypeptides. However, in immunoblotstudieswith two preparations platelets to fibronectin(FN), vitronectin(VN), or von Willebrand factor, of polyclonal anti-human-o/B, the avian FNRB, cross reacted with other ligands which lack the y400-41 1 sequence but also bind to platelets the anti-human-B1 fraction of the antisera, while the avian FNR 165 via cllb/p3. At about 1 mM, a slight inhibtion of adhesion to VN was observed. Thus the inhibitory effects of kD cL, subunit from the different tissues did not show cross- DPPRFVRPLQ may be specific for reactivity; this indicates that avian is antigenically different from FG, and if the binding of FG via its y400-41 1 sequence is limited to a, allb/p3, then the peptide may be platelet specific. This the human counterpart. The 155 kD a5a subunit yiekled a single possibility is supported by the observations that DPPRFVRPLQdid not inhibit theaS/fI1 160 kd polypeptide upon reduction, indicating that this is FN- mediated adhesion ofplatelets to FN, nor, in contrast to GRGDSP, the specific but is-different from or a, may be a novel FN- and a, as, gastniation of Rana pipiens embryos. Thus, this peptide may provide a RGD-specific a subunit that associates with B, subunit. (a,,?) structural rationale for the design of a potent, platelet specific anti- thrombotic agent. Monday. Cell Receptors and Extracellular Matrix I (1040-1042) 179a

1040 1041 IDENTIFICATION OF A COMMON HYALURONAN BINDING MOTIF IN THE RECEPTOR CANDIDATES FOR ENDOTHELLAL CELL BINDING HYALURONAN-BINDING PROTEINS RHAMOS, CD44 AND LINK PROTEIN PEPTIDES FROM THREE SEPARATE DOMAINS OF ((Yang, B., B.L. Yang, R.C. Savani and Z.A. Turley)) Inst. of THROMBOSPONDIN-1. ((W. A. Frazier, M. B. Finn and A.-G. Gao)) Cell aiology and Dept. of Pediatrics, University of Manitoba, 100 Olivia Street, Winnipeg, Manitoba, Canada, R3 OV9 Department of Biochemistry and Molecular Biophysics, Washington We have previously identified two hyaluronan (HA) binding University School of Medicine, St. Louis, MO 63110. domains in the HA receptor RHAIM that occur near the carboxyl terminus. We show here that these two HA binding domains are Thrombospondin-l (TS1) plays important roles in the regulation of the only HA binding regLons in RHAMa, and that they contribute endothelial cell (EC) phenotype. In particular, TS1 has been shown to approximately equal, to th? 3SA binding. Mutation of domain II demonstrates that X and R 3, spaced 7 amino acids apart, are inhibit angiogenesis in vivo (Good et al, Proc. Natl. Acad. Sci. USA critlcal for HA bindlng activity. Domain I contains two sets of 87:6624, 1990), and peptides from two domains ofTS 1, the procollagen baslc amlno acids, also spaced 7 residues apart, and mutation like region and the type 1 or "malaria-like" repeats, are also angiostatic in of these resLdues reduced their bindlng to HA-Sepharose. These vivo and inhibit EC migration in vitro (Tolsma et al., J. Cell Biol. 122:497, results suggested that basic amlno acid that flank a 7 amino 1993). We have extended these studies by examining the attachment of acid stretch (iL.. B(X confer HA-binding actlvity. To )B) types of TS1. The EC include assess whether this molf predLcts HA binding in the intact three ofEC to 15 peptides from 4 domains RHAMN proteLn, we mutated all basic amino acids in domains I calf pulmonary artery (CPAE), a large vessel EC, bovine adrenal capillary and II that form part of these motifs. The altered RHAMN EC (BACEC) and polyoma middle t transformed mouse brain EC proteln dld not bind HA, confirming the critical nature of the (bEND.3). A 25mer (LRRPPLCYHNGVQYRNNEEWTVDS) and its basic amino acids and their spacing to binding. A specific shorter homologs from the procollagen-like domain, a tryptophan-rich requirement for arg inepr lyu,Jine residues was identified peptide from the type 1 repeats (KQDGGWSHWSPWSSC) and two since mutation of , R4, KW to histidine residues abolished binding, Clustering of basic amino acids enhanced HA binding peptides identified as potent cell binding sites from the C terminal domain, activity while occurrence of acidic residues between the basic (7N3 = FIRVVMYEGKK and 4N1 = RFYVVMWKQVTQS) all bound amino acids reduced blnding. The B(X )B motif was found in all EC well when adsorbed to plastic. The peptides from the type 1 repeats of HA-binding proteins. Recombinant techniques were used to TS surrounding the CSVTCG sequences bound cells only weakly, while generate chimeric proteins containing either the B(X7)B motifs three heparin binding peptides from the amino terninal domain of TS1 present in CD44 or link protein. All chimeric proteins containing the motif bound NA in traneblot analyses. Site- were all inactive in binding EC. Iodinated active peptides were used with directed mutations of these motifs in CD44 abolished HA crosslinking reagents, to identify receptor candidate proteins on EC. binding. These results predict that the motif of B(X7)B as a Peptides 7N3 and 4N1 specifically label the same three proteins, while the binding requirment for HA in RHAM5, CD44 and vlink protein. procollagen-like and tryptophan rich peptides each specifically label Research was supported by an NCIC grant to E.T. different, single protein bands. (Supported by HL14147 and Monsanto Co.)

1042 CELL AND HEPARIN BINDING IN THE DISTAL LONG ARM OF LAMININ: IDENTIFICATION OF ACTIVE AND CRYPTIC SITES WITH RECOMBI- NANT AND HYBRID GLYCOPROTEIN. ((U. Sungt, J.J. O'Reart and P.D. Yurchencotl). Dept. of Pathologyt and Molecular Genetics & Microbiol- ogyt, Robert Wood Johnson Medical School, Piscataway, NJ, 08854.

The long arm of basement membrane laminin consists of A, Bi and B2 chains joined in a triple coiled-coil. In order to analyze its functions, we have expressed the distal moiety of the mouse laminin A chain extending from the middle of the long arm to the C-terminus. This glycoprotein (rAiG), secreted by Sf9 insect cells infected with recombinant baculovi- rus, was intercalated in vitro into the corresponding Bi and B2 chain segments isolated from laminin fragment E8. The resulting hybrid mole- cule (B-rAiG) possessed a structure similar to the laminin long arm as judged by electron microscopy and limited proteolysis. By joining rAiG to the B chains, the affinity of the G domain for heparin decreased from a level observed with rAiG and rG (recombinant G domain) to a level simi- lar to native protein. HT1080 cells adhered to fragment E8, rAiG, and B- rAiG, poorly to rG, and not to denatured E8/B-rAiG, the separate A and B chain moieties of E8 or a mixture of rG and B chains. Furthermore, cell adhesion to E8 and B-rAiG, in contrast to rAiG, was inhibited with monoclonal antibodies specific for the a6 and 01 integrin chains. Thus subunit intercalation (a) restored the native a601 integrin binding site, (b) inactivated a cryptic cell binding activity in the A chain, and (c) inhibited a heparin binding site in the proximal regions of G domain. These data provide evidence that the multi-subunit structure of laminin possesses conformationally-dependent activities not present in isolated subunits. Extracellular Matrix and Cell Behavior II (1043-1044)

1043 1044 AGE-DEPENDENT CHANGES IN ANGIOGENESIS ARE ASSOCIATED MUTUAL INFLUENCE ON COLLAGEN SYNTHESIS AND PROLIFERATION BY WITH POOR GROWTH OF THE EHS TUMOR IN C57 MICE. ((R. Pili, Y. Guo, TGF-B1 AND PDGF-A/B IN ARTERIAL SMOOTH MUSCLE CELLS. J. Chang, T. Novilck, H. NakanIshi, C. Yang, G. R. Martin, and A. Passaniti)) ((U. Falken and H. Robenek)) Inst. for Arteriosclerosis Research, University of Minater, Laboratory of Biological Chemistry, National Institute on Aging, Baltimore, MD Domagkstr. 3,48149 MOnster, Germany. 21224. We examined synergistic effects of PDGF-A/B and TGF-B1 on protein synthesis, collagen Important in the We have utilized a rodent model to Investigate the mechanisms production and proliferation capacity in enzymatically isolated SMCs from the aortic n)edis reduced rate of growth of tumors in old hosts. The transplantable Englebreth- of growing SMCs was determined by ("H)- Holm-Swarm tumor (EHS) was found to form 3 to 4-fold larger tumors In young swine. Proliferation of subconfluent young as well as adult CS7BL mice compared to old mice of the same strain. thymidine uptake, cell counting and cell cycle determinations. To measure DNA synthesis, Rapid tumor growth was restored upon transplantation of the tumor into young SMCs were incubated for 24 h with 5 ng/ml PDGF-A/B togehter with various TGF-Bl animals suggesting that host factors rather than cell selection determine the rate qqncentrations (0.01 ng/ml up to 1 ng/ml) in medium containing 05 % serum and W,Ci/ml of tumor growth. Also, the rate of DNA synthesis of old tumor tissue in organ (H)-thymidine. Samples were determined as TCA insoluble products in a liquid culture was increased after 8 hours and reached levels seen in tumor tissue from scintillation counter. To measure cell repication, SMCs were incubated with growth factors young animals consistent with the loss of a host inhibitor. The histology of for 48 h without radioactive labeling and the cells were counted. Cell cycle determinations tumors from old animals differed strikingly from tumors obtained from adult of propidium iodide labeld nuclei were performed vith a FACScan after an incubation animals with a 3-fold Increase in the ratio of extracellular matrix to tumor cells. period with growth factors of 24 h. TGF-B1 downregulates in a dose-dependent manner the numerous vessels In addition, tumors from adult animals exhibited Infiltrating PDGF-A/B induced increase of SMC proliferation, yielding a maximal 2.9-fold inhibition of from old animals contained fewer vessels which were usually much while tumors DNA synthesis corresponding to a reduction of cells being in S-phase from 233% to 9.5% larger and were dilated. Linomide, a potent anti-angiogenic agent, dramatically a an in Inhibited tumor growth in both young and old mice indicating that the EHS and reduction of the cell numbers to amount found controls. as and tumor depends on an adequate blood supply for rapid growth. Using an To measure protein/collagen synthesis, SMCs were cultured confluent monolayers Independent angiogenesis assay, young animals supported a greater and more preincubated for 24 h with 5 ng/ml PDGF-A/B in combination with 1 ng/ml TGF-B1 in rapid angiogenic response compared to old animals. Extracts from tumors medium containing 17D serum. Cells were further incubated with growth factors for 24 h in growing in old animals failed to support in vitro endothelial cell differentiation the presence of (1 C)-proline. For RNA preparation, cells were isolated after the and these aged extracts also Inhibited tumor cell growth In vitro while extracts preincubation period. TGF-B1 induced a 1.-fold increase in total proteins produced, and in from tumors growing in young animals promoted iL Incidence and subsequent addition a specific 1.9-fold enhancement in the proportion of collagen from 10% to 19%. growth of either EHS tumor cells or human prostate tumor cells transplanted to The corresponding lvel of collagen type I mRNA was also found being 1.-fold increased studies nude mice was Inhibited by tumor extracts from aged C57 mice. These TGF-B1. PDGF-A/B alone had no influence on the increased synthesis of total proteins, the old host slow the rate of and by suggest that factors produced by angiogenesis in combination with TGF-B1, the specifically enhanced collagen proportion and of tumor Such factors could also modulate the repair and regeneration however, growth. was to of normal tissue with age. mRNA value reduced those presented by control cells. 180a Extracellular Matrix and Cell Behavior II (1045-1050). Monday

1045 1046 EXTRACELLULAR MATRIX PROTEINS CAN REGULATE THE EGF RECEPTOR ACTIVATION IS REQUIRED FOR THE ACTIVATION AND VIABILITY OF EOSINOPHILS. ((A. Tourkin, E.C. FORMATION OF ORGANTYPIC STRUCTURES BY NORMAL LeRoy and S. Hoffman)) Medical University of South Carolina, HUMAN BREAST EPITHELIAL CELLS GROWN ON Charleston, SC 29425. RECONSTITUTED BASEMENT MEMBRANE. ((L.K. Opresko, P.M. Burke, and H.S. Wiley)) Department of Pathology, University During migration to sites of inflammation, eosinophils (Eo) pass through of Utah Medical Center, Salt Lake City UT 84132. two regions rich in extracellular matrix (ECM) proteins: basement membrane and connective tissue stroma. To evaluate what effect these ECM proteins might have on Eo behavior we examined the interaction of Normal human mammary epithelial cells (HMEC) will form a Eo with ECM proteins. We found that control Eo (from healthy monolayer when grown on plastic, but will organze into individuals) adhere no better to substrates coated with laminin (LM), glandular-like three-dimensional structures when cultured on fibronectin (FN), cytotactin (CT) or collagen types I or IV (CoIV) than reconstituted basement membrane material derived from the they do to human serum albumin (HSA). In contrast Eo activated in..itro Englebreth-Holm-Swarm (EHS) murine sarcoma. To investigate with some cytokines (IL-S, IL-3, GM-CSF) or il vivo in patients with the role of the EGF receptor in this process, we utilized normal 184 eosinophilia bind well to LM, FN and CoIV. LM is the most avid ligand, HMEC as well as the immortalized counterpart 184A1 grown in 43% of input cell bind to a substrate 200 of a similar bearing fm/cm2 LMK serum-free medium. When placed on EHS matrix, these cells level of adhesion to FN required 30 times as much adsorbed protein. CT rapidly assembled into glandular-like structures inhibits the adhesion of Eo to LM. Antibody inhibition stadies suggest (<24hrs). Blocking EGF receptor occupancy by the addition of monoclonal that the integrin is the predominant LM receptor on Eo. Flow antagonistic a6p, antibody 528 prevented this process. The 528 antibody also cytometry showed that levels of co,s integrins were not changed after activation. The effect of ECM proteins on Eo behavior was also completely blocked cell division in either the presence or absence of examinated. LM coated substrates promote viability and activation al EHS matrix. We found that HMEC cells express extremely high vitro with no added cytokine; equally adhesive FN-coated substrate had levels of EGF receptors (between 1-3 million/cell) due to extensive much less effect. Conditions that inhibit adhesion to LM (anti-integrin receptor recycling. In addition, these cells also produce high levels Abs in the medium or CT on the substrate) and Ab against IL-3 and GM- of TGF-alpha. However, occupancy of only a small fraction of the CSF inhibited the promotion of Eo viability by LM. These observations EGF receptors was necessary to support growth of HMEC and suggest that the interaction of leukocytes and ECM proteins may be stimulate formation of glandular-like structures. Our data indicate important for their ability to regulate the viability, cytokine production, that EGF is not only involved in the regulation of HMEC and activation of Eo. mitogenesis, but also in the formation of differentiated structures.

1047 1048 REGULATION OF DIFFERENTIATION AND EXTRACELLULAR MATRIX (ECM) CONSTRUCTION AND APPUCATION OF ENVIRONMENTS FOR IN VITRO EN- GENE EXPRESSION IN RAT TRACHEAL EPITHELIAL (RTE) CELLS BY DOTHELIAL CELL GROWTH AND DIFFERENTIATION. ((B.J. Del Buono)) Colabora- SUBSTRATUM. ((E.A. Davenport and P. Nettesheim)) Pulmonary tive Biomedical Products, Becton DIWnson and Company, Bedford, MA 01730. Pathobiokxgy, NIEHS, Research Triangle Park, NC 27709. The extracellular matrix (ECM), growth factors, and cells all interact to define the Primary RTE cells differentiate into a mucociliary epithelium when grown cellular mlr enronment These Iteractons hi turn, play a crucil role hi regulating on collagen (Col I) gel-coated membranes in air liquid interface cultures celular physioogy both k_n,_ and Jo.k_p. In an elfort to consruc Joy r environ- (In Vitro Cell. Dev. Bio., 29A:481, 1993). In addition, in submerged RTE mat proIding teopstmi conditions to regula the p idogyd ndoxlW cces cultures on plastic that do not undergo mucociliary differentiation, we have (EC), various aIon d ECM and growth bctors w examnd abli- observed high levels of a-1 collagen IV (col IV) and fibronectin (FN) ties to Induce EC growth or differentiatIon. EC from several sources (species and expression compared to differentiated cultures on Col gel-coated vesel types) were seededito stndard tissue culture fis, or ito BlooatR flasks membranes. To understand the effects of substrata on RTE differentiation treated with collagen 1, fibronectin, or laminin, and were grown In a low-serum and ECM gene expression, RTE cells were seeded on uncoated or Col gel- medium containing varous growth factors Including bFGF, EGF, or ECGS/ECGF. coated polycarbonate membranes and growth, differentiation, and ECM gene Cells grown on coliagen for five to seven days demonstrated a two- to six-fold expression were monitored. Only minor differences were observed between greater increase In oell number compared to those seeded Into standard flasks or the two conditions in regard to cell attachment frequency, growth rate, and onto laminin or fibronectin. This mitogenic effect of coliagen was maximal when plateau cell density. However, in early cultures, numerous small colonies EGF, ECGS, and heparin were Included In the optimized low-serum medium. For were seen on giemsa-stained uncoated membranes while on Col gecoated Induction of dlfferentlatlon. however, the optimal ECM waseither fibronectin or membranes, cells formed one central contiguous sheet. Biochemical evidence Basement Membrane Matrlgei. EC grown on permeable membranes treated with of mucous secretion was observed on both uncoated and Col gekcoated thee EON compont but not on thoeetrseat with colagen 1, formed conluert membranes but the secretory (mucous) phenotype developed later on monolayers that demonstrated barrier function (determined by measurement ot uncoated membranes. Ciliated cells were detected readily only on ColI gel- electricl itance dye diffuson) and displayed markers EC coated membranes. Col IV and FN were expressed at higher levels in early differentiation (vWF expression; AcLDL binding). These differentiated monolayers RTE cultures on uncoated membranes than in late cultures on uncoated proved usefti In a variety applications. Including the study transendotheial membranes and both early and late CoI ge-coated membranes. These data leukocyte trafficking. Interactions betwen components the extraceilular matrix suggest that Col I gel is not essential for growth or secretory cell and growth atorsthus help to determine the fated EC (growth or diri), differentiation of RTE cells; however, it accelerates appearance of the and bnowdge and epoiadon thm eractons allow the secretory phenotype. In addition, expression of some ECM genes negatively mizedILn _)dP environments designed to Induce eithr growth or differentiation correlates with secretory differentiation. endotheil cells.

1049 1050 TENASCIN-MEDIATED LOSS OF TISSUE-SPECIFIC GENE EXPRESSION A NOVEL ROLE FOR THYMOSIN 84: AMATRIGEL INDUCED GENE IN MAMMARY EPITHELLAL CELLS. ((Peter Lloyd Jones*, Ruth Chiquet- INVOLVED IN ENDOTHELIAL CELL DIFFERENTIATION ((D.S. Ehrismann+ and Mina J. Bissell*))*Life Science Division, Lawrence Berkeley Grant, J.L. Kinsella#, M.C. Kibbey, S. LaFlamme, P. D. Burbelo, A. L. Laboratory, University of California, Berkeley, CA 94720, and+Friedrich Goldstein*, and H.K. Klcinman.)) Laboratory of Developmental Biology, Miescher-Institut, CH-4002 Basel, Switzerland. NIDR, NIH, Bethesda, MD 20892; # Laboratory of Cardiovascular Science, GRC, NIA, Baltimore, MD; and * Dept. of Biochemistry and The extracellular matrix (ECM) plays a key role in regulating the Molecular Biology, George Washington University Medical School, differentiated state of mammary epithelial cells. Tenascin is a large, oligorneric Washington D.C. ECM glycoprotein that is believed to play an important role in morphogenesis, cell migration and tissue repair. It can interact with a number of ECM We performed differential cDNA hybridization using RNA from cells at 4 molecules and possesses both adhesive and anti-adhesive domains. We have hours on plastic and Matrigel, to define the early genes involved in the examined the expression of tenascin in the mammary gland in vivo and have differentiation of shown that it decreases during pregnancy and lactation, but is re-expressed morphological endotheral cellsinto capillary-like tubes on during involution. In culture, the onset and maintainence of the lactational Matrigel. One clone, thymosin 84, exhibited a 5-fold increase in cells on phenotype by SCp2 cells, a mammary epithelial cell line, relies upon the Matdgel. Immlunostaining localized thymosin 84 to the endothelial cell continued presence of both lactogenic and the laminin-rich EHS comprising the tubes, and in vivo thymosin B4 was present mainly in biomatrix (Desprez et al., MoL Cell. Diff. 1993, 1:99-110). Under these growing and mature vessels. HUVECs, transfected with thymosin 84 differentiating conditions, SCp2 cells exhibit an absolute requirement for cell showed increased attachment to and spreading on plastic, laminin and clustering in order to synthesize the milk protein beta-casein. Given both the collagen IV, and an accelerated rate of capillary-like tube formation on adhesive and anti-adhesive properties of tenascin together with its pattern of Matrigel. Finally, an antisense oligo to thymosin 84 inhibited tube expression during adult mammary gland development in vivo, we have studied formation on Matrigel. Thymosin 84, previously thought to be a thymic the effect of tenascin upon the phenotype of SCp2 cells cultured in lactational , now appears tobe a product and regulator of differentiating the presence of lactogenic hormones and We have shown that tenascin EHS. endothelial cells and is likely significantin vessel formation. can inhibit beta-casein expression in a dose-dependent manner. In addition, the effect upon milk protein synthesis is accompanied by a reduction in cell clustering, which m turn may reflect alterations in the cytoskeleton; these include both a dissolution of actin filaments as well as the induction of a 4OkD cytoskeletal protein. Taken together, these and other data argue that the expression of tenascin is incompatible with the lactational phenotype and that this ECM molecule may play a decisive role in regulating the differentiated phenotype of the mammary epithelium. Monday. Extracellular Matrix and Cell Behavior II (1051-1056) 181a

1051 1052 EFFECTS OF TRANSFORMING GROWTH FACTOR-01 ON THE ADHESION, MIGRATION ENDOTHELIAL CELL RETRACTION: DIFFERENTIAL RESPONSE IS AND INVASION OF HIGH AND LOW METASTATIC MURINE MELANOMA CELLS. ((K. MODULATED BY SPECIFIC MATRIX PEPTIDES. ((J.M. Onoda, S.S. Kantak, Ellingson 1, J.B. McCarthy2, and L.A. Repesh1)) Department of Anatomy and Cell and C.A. Diglio)) The Gershenson Radiation Oncology Center and the Biology, University of Minneaota, Duluth, MN1, and Department of Lab Medicine and Departments of Radiation Oncology and Pathology, Wayne State University Pathology, Univeraity of Minnesota, Minneapolis, MN2. School of Medicine, Detroit, Ml 48201. P Transforming growth factor (TGF-01), a regulator of growth and differentiation, We determined the effects of low dose radiation ( 150 cGy) on pulmonary is Is present in a wide variety of normal and malignant cells. TGF-Pl especially high microvascular endothellal cell (PMEC) morphology and F-actin organization. We in platelets and bone and is found circulating in the aerum where it may influence observed dose- and time-dependent reversible radiation induced injuries to tumor cell/basement membrane interactions. The effects of TGF-01 on the adhesion PMEC monolayers characterized by retraction (loss of cell-cell contact) migration, and invasion of high (M2) and low (10) metastatic clones of the K1735 mediated by cytoskeletal reorganization. Radiation induced reorganization of murine melanoma cell line was inveatigated. Adhesion was measured In microtiter F-actin microfilament stress fibers was observed > 30 minutes post irradiation wells coated with fibronectin following treatment with TGF-p1 (0.0, 0.1, 1.0, 10 and correlated positively with changes in PMEC morphology. Cells recovered > ng/ml) for 24 hours. The adhesion of M2s to fibronectin was inhibited, whereas thE to form contact inhbited monolayers 24 hours post irradiation; concomitantly, adhesion of 10's to fibronectin-coated substrata was unaffected. The migration of the depolymerized microfilaments organized to their pre-irradiated state as tumor cells in response to fibronectin through polycarbonate filters in microBoyden microfilament stress fibers arrayed parallel to the boundaries of adjacent chambers was measured following pretreatment with TGF-Pl for 24 hours (0.0, contact-inhibited cells. Initial studies were performed on PMEC derived 0.1, 1.0, 10 ng/ml). Migration increased 1.5 fold in the M2 cells and 2.3 fold in the substrata. In later studies, we examined endothelial retraction of cells plated on clone 10'a. Finally, invasion through matrigel-coated polycarbonate filters was specific extracellular matrix peptides. We observed that cells plated on type IV measured in the presence (10 ng/ml) or absence of TGF-f1 for 24 hours. Invasion coliagen were nore resistant to radiation-induced F-actin reorganization and was increased 2.5 fold in the M2 cells and 1.8 fold in the clone 10's. Collectively, demonstrated less cellular retraction in comparison to cells plated on laminin, these results have demonstrated the ability of TGF-p1 to modulate the activity of a which demonstrated increased sensitivity to radiation-induced F-actin low and high metastatic clone in vitro. Importantly, TGF-P1 alters the invasive and reorganization and retraction. Our data suggest that the quality of cell-cell motile properties of a low metastatic clone to more closely resemble the malignant adhesion and cell-substrata adhesion modulate the response of endothelial phenotype of a highly metastatic clone. Further characterization of how TGF-p1 and monolayers to extemal agonists (i.e., radiation). Moreover, these differential responses may other potential autocrine-paracrine factors regulate tumor cell/basement membrane help explain experimental observations by different at may interactions could potentially Identify specific targets for therapeutic intervention investigators which, times, be at variance. These studies were by Gershenson in the treatment of cancer. (Supported by Ladies' Auxiliary to the Veterans of supported PHS CA50466 and the Radiation Oncology Center. Foreign Wars 16221and NIH CA 439242).

1053 1054 INTERLEUKIN 8, BUT NOT TNF OR FMLP, STIMULATES GROWTH CONE ADHESION TO SUBSTRATUM-BOUND LAMININ. ((W.A. NEUTROPHIL MIGRATION THROUGH FIBRIN GELS. ((J.D. Loike, J. Thomas, J. Bardsley and A. J. Castellino)) Department of Natural Science, El Khoury, L. Cao, I.J.D. Lindley S.C. Silverstein)) Department of Colby-Sawyer College, New London, NH 03257 Physiology, Columbia University College of Physicians and Surgeons, New York, NY 10032 Sandoz Research Institute, Vienna, Austria. It has been suggested that growth cone/substratum adhesion adhesion might play an instructive role in regulating neurite elongation, but tests of that hypothesis have been inconclusive. Here we present results demonstrating a such as Chemoattractants N-formyl-methionyl-leucyl-phenylalanine (fMLP), correlation between the rate of neurite elongation across a substratum and tumor necrosis factor-a (TNF) and interleukin 8 (IL-8) stimulate human adhesion of the growth cone to that substratum Neurites from dorsal root polymorphonuclear leukocyte (PMN) migration through a variety of extracellular ganglia of 8-day chick embryos were cultured under serum-free conditions matrices. We report that PMN migrationthrough a fibrin gel depends upon the in Nunc 4-well plates coated with different concentrations of laminin (LN). chemoatrca. PMN migration was examined using Becton-Dickinsen tissue Cultures maintained on each substratum were photographed, subjected to a culture inserts (pore size Siam) that were pre-coated with extracellular matrix standard gyratory shear, and then rephotographed. Resulting images were analyzed for the number of originally adherent growth cones proteins. PMNs were added to the upper chamber of the inserts and allowed to remaining attached after exposure to the shear force employed. The mean rate of migrate for up to 8 hrs at 370 C in response to the addition of chemoattractants neurite elongation was also determined on each substratum as previously to the bottom chamber. In the absence of a chemoatractant or in response to described(Thomasetal. (1990) Deveopment 110: 1101-1114). The TNF (3 x 10- M) orfMLP (10- M), fewer than 0.6% of the PMNs migrated results indicate that for the LN concentrations tested, a relatively high rate throughinserts coated with fibrin. In contrast, at least 15-20% of the PMNs of neurite elongation was associated with a more tenacious attachment of the migrated across a fibrin gel in response to IL-8 (2.5 10x M). The effect of IL- growth cone to the substratum. Moreover, exposure of the cultures tI 8 on PMN migration was dose dependent. When IL-8 was added together with reagents (a-lactalbumin, glunac, chitotriose) known to inhibit activity of p the cells in the upper chamber of the inserts no migration was observed, 1,4 galactosyltransferase (galtase) and reduce the mean rate of neurite indicating that a chemical gradient was required. Control experiments elongation increased the percentage of growth cones removed by the standard showed gyratory shear. These results indicate that growth cone/substratum that in response to TNF, IL-8 orfMLP, PMNs readily migratedthrough inserts adhesion may help determine the rate of neurite elongation across LN-coated coated with fibrinogen, fibronectin or reconstituted basement membrane substrata and may itself be regulated by the activity of cell surface galtase. (matrigel). These results sugest that the nature of both the extracellular matrix The findings lend support to the hypothesis that changes in growth and the chemot regulate PMN migration. cone/substratum adhesion can provide directional cues to elongating axons under appropriate circumstances.

1055 1056 DOMAIN-SPECIFIC REGULATION OF NEURITE OUTGROWTH- AND NEURON GLYCOSYL GROUPS OF LAMININ ARE NOT IMPORTANT FOR MIGRATION-PROMOTING ACTIVITIES OF LAMININ. ((A.D. Lander1, M.R. INTERACTIONS OF RODENT MYOBLASTS WITH THIS GLYCOPROTEIN Campanerol, P.D. Yurchenco2and A.L.Casof3)) 1Departments of Brain and SUBSTRATE.((T.Y. Kostrominova and ML. Tanzer)) Department of BioStructure Cognitive Sciences and Biology, MIT, Cambridge, MA 02139, 2Department of and Function, School of Dental Medicine, University of Connecticut Health Center, Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 08854, and Farmington, CT 06030-3705. 3Depamnt of Biogical Scienc, University of ws, lows Ciy,IA 52242. Larninin (Ln) is a major glycoprotein of basement membranes. It plays an The extracelluar matrbi protein amninn (LN)ia believed to playimportant roles in important role in growth, locomotion and differentiation of manycell types and cell/ neuraldevelpn, guiding growing axons andmiigtingneurons to their targets. Ln interactions are important for myogenesis. The role of Ln glycosyladon in its In vitro, moat cases of neurons respond to substratum-bound LN by extending biological functionsis just emerging. We reporthere the effects of the carbohydate of Ln on myoblastadhesion, neurites; a fewalso undego el body migration. In the course of studis ainWd at moiedes spreading and differendation. Unglycosylated from tunicamycin-treated mouse cell was mapping functional tes on LN, wediecovered what appearstobe a new, regulated lamninin cultures of line, M1536 B3, used is is in the experiments. Glycosylated laminin from EHS tumor and from M1563 B3 ceUs acvity ofthe LNmolcule. This acivity not normally detected in LN that bound a to gla ortisue cukture plticsubstrata, but it is induced by treatment of LN served as control. Cell binding experiments with C2C12 mouse myoblasts and L6 bt with affinity purifiedantibod s directedagainstits El'or P1 domains. it rat myoblasts showedthat boLh types of cells preferrd a laminin-coated surface, to an uncoated type then readily deteoced by at lt two types ofneurons: Enbryonic mouse olfactory compared plastic surface (nontissue culture-treated). Neither cell receptor neurons normally migate in respons to substratum-bound LN, but their distinguished between glycosylated andunglycosylased Ln substrates. Pretreatment of migration is 2-3 fold greater on lamininsubstrata pretreated witheither of these glycosylated and unglycosylated laninin substrates with Elderberry or Concanavalin A lectins did not decrease myoblast attachment and spreading. Both lectins, antibodies. In the presence of antibodies that block the function ofa6 integrins, when migration on untreated LN isbbcked,butmigration onanti-P'-treated LN persists. adsorbed to the plastic surface, before binding experiments, enhanced myoblast Lateembryonicrat, mouse or chicken retinal neurons normaly extend fewif any spreading. In contrasttbe same lectinsimpaired cell spreading on both glycosylated and unglycosylated Ln substrates when lectins were added in solution with the ceUs. neuritesin reponse to LN substrata, but do so profusely when the substrata ae treated with anti-El' or anti-PI'. The response is integrin-dependent, but Both glycosylated and unglycosylated forns of laminin promoted myoblast growth ae 01 and differentiation. In contrast, on uncoated surfaces involves neither nor RGD-senative integns.Inrterestingly, the responses of myoblasts plastic grew very slowly and did not further differentiate. Theseresults indicate that although both retinal and olfatory neurons to anti-Pli-treaed LN are blocked by 'antbodies d myoblasts requirethe presence of laminin for spreading, growth and differentiadon on to domain ES, apart LN quite distant(>40 nm) from where aniti-El' and -P1' a proprietary plastic surface,Ln carbohydrates are not implicated in those cellular antibodies bind. In addition, isolated ES posesses the activity that intact LN responses. by grant only acquires when treated with anti-El' or -Pl'. These data suggest that induction (Supported NIH AR17220). of neurite outgrowth-promoting and migration-stimulatingactiviy by anti-El' and anti-PI' antibodies Is probably not due to effects on locl protein coriormation of LN, but rather due to bocde an inhibitory function resiig in the P1' domain. 182a Extracellular Matrix and Cell Behavior II (1057-1062). Monday

1057 1058 INVOLVEMENT OF a 2 INTEGRIN DURING THE ACIDIC FGF INDUCED FUNCTIONAL ROLE OF LFA-1, VLA-6, AND CD44 FOR T CELL SCATTERING OF THE RAT BLADDER CELL LINE NBT-I. ((A.M. Valles, G. LOCOMOTION IN 3D COLLAGEN GELS. Tarone°, J.P. Thiery, and B. Boyer)) Laboratoire de (P. Friedl', P.B. Noble2, and K. S. Zanker')) ' Naturwissenschaftliche Physiopathologie du D6veloppement, CNRS-URA 1337, Ecole Normale Fakuftat, Universitat Witten/ Herdecke, Germany, 2 Facuity of Dentistry, Superieure, 46 rue d'Ulm, 75230 Paris 05 France and °Dipartimento McGill University, Montreal, P0, Canada. di Genetica, Biologia e Chimica Medica, Universita di Torino, via Santena 5bis 10126, Torino, Italy. (Spon. by C. Rebut-Bonneton) Cell adhesion molecules play a major role in the interaction of a wide range of cell types with the surrounding extracellular matrix (ECM). We Cell adhesion receptors of the integrin family are known to investigated the effects of monoclonal antibodies (mAb) binding to LFA-1 participate in the maintenance of cell anchorage and cell (CD1 1a, mAb 25.3.1.), VLA-6 (or, mAb GoH3), and CD44 (mAb J-173) migration by mediating interactions between cells and on immunomagnetically isolated human CD4 and CD8+ lymphocytes. extracellular matrix molecules (ECM) and in some cases, between The spontaneous and rlL-8 induced migration in 3D collagen gels was neighboring cells. The rat bladder carcinoma cell line NBT-II examined. The paths of randomly selected cells were digitized and undergoes an epithelial to mesenchymal transformation (EMT) quantitatively analysed. when grown on collagen, an ECM molecule, or after addition of Incubation of CD4 and CD8+ cells with VLA-6 and LFA-1 mAb did not growth factors like acidic FGF: the epithelial cells dissociate, begin after the spontaneous locomotory activity. However, subsequent to migrate actively and acquire a fibroblastic-like appearance. activation with rIL-8 (20 ng/ml) was almost completely inhibited indicating During this period, an increase in expression of a2 integrin is that functionally intact VLA-6 and LFA-1 receptors are required for rlL-8 observed when cells are plated on collagen or when induced with induced migration. aFGF. A more striking increase in a2 expression Is seen when both In contrast to LFA-1 and VLA-6 mAb, CD44 mAb induced a rapid and inducers are present which correlates with an enhancement in the transient increase in cells locomoting (30 to 40%; CD4+ > CD8+), which speed of locomotion. In agreement with these results, a2 integrin is significantly declined after 20 to 30 min below control levels. Additional the major collagen-binding species in NBT-II cells. To clarify the stimulation with rIL-8 or PMA (50 tg/ml) restored the capacity to function of a 2 integrin during the EMT of NBT-II cells, we locomote indicating that the locomotion persewas not impeded by CD44 introduced the full length cDNA clone for the human a 2 chain. In mAb. Incubation with irrelevant mAb (IgG,, isotypic control) did not affect a2 -transfected cells, blocking a 2 antibodies inhibited cell lymphocyte behavior under any condition investi9ated. scattering induced by acidic FGF as observed by time-lapse The data suggest that VLA-6 and LFA-1 act in a similar manner whereas cinematography. They also inhibited wound repair induced by CD44 may comprise a functionally distinct role in lymphocyte locomotion. aFGF. These results support a role for a2 in cell motility during the process of EMT.

1059 1060 THROMBOSPONDIN PROMOTES CHEMOTAXIS AND HAPTOTAXIS OF VASCULAR SMOOTH MUSCLE CELL MIGRATION: THROMBOSPONDIN IS HUMAN PERIPHERAL BLOOD MONOCYTES. ((P.J. Mansfield and SJ. AN AUTOCRINE MOTILITY FACTOR ESSENTIAL FOR MOTILITY IN Suchard)) Deparlnent of Pediatrics, University of Michigan, Ann Arbor, MI 48109. RESPONSE TO PDGF. ((R. YabkowitZI, P.J. Mansfield2, U.S. Ryan3, and S.J. Suchard2)) Depts. of Pathologyl and Pediatrics2, Univ. of Michigan Medical School, The presence of extracellular matrix proteins such as thrombospondin (TSP) at sites Ann Arbor, Ml 48109 and Dcpt. of Surgery3, Washington Univ. School of Medicine, of inflammation or injury may promote monocyte (MO) migration to these sites and St. Louis, MO 63110. their eventual differentiation to tissue macrophages. Previously, we have shown that TSP promotes both neutrophil adhesion and migration. To examine the effect of TSP The development of atherosclerotic lesions and plaques is accompanied by the on MO motility, we conducted chemotaxis assays in modified Boyden chambers. TSP inappropriate migration and proliferation of smooth muscle cells (SMC) to form a was chemotactic for MOs, with a maximal rsponse at 200-500 nM TSP. mAb C6.7, neointimal layer in the vascular wall. The extracellular matrix protein, against the distal COOH terminus of TSP, inhibited chemotaxis dem aig thuombospondin (TSP), is a primary rsponse gen in PDGF-stimulated SMC. We specificity. Consistent with the mAb data, the COOH-terminal 140-kD proteolytic have examined the role of TSP in SMC chemotaxis and found that TSP is a potent fragment of TSP was chemotactic for MOs, while the NH2-tenninal heparin binding stimulator of motility. Inhibition of SMC chemotaxis by the anti-TSP mAb C6.7 domain (HBD) had no effecL A synthetic peptide containing the sequence CSVT, indicated that this response was specific and mediated by the distal COOH tenninus. derived from the type I repeats of the 140-kD fragment, was also chemotactic. Thus An at-containing receptor but not a3 or a,0s, was implicated in mediating SMC two sites on the COOH terminus may be capable of stimulating MO chemotaxis. migration to TSP. Based on the similar kinetics of TSP and PDGF expression at sites Pertussis toxin (PT), but not cholera toxin (Cr), completely inhibited TSP-mediated of vascular injury, the effect of suboptimal levels of TSP on SMC migration to PDGF chemotaxis, suggesting the involvement of G-proteins in tWs process. TSP bound to was also evaluated. In the presence of low concentrations of TSP, SMC chemotaxis to polycarbonate filters also stimulated the directed migration (aptotaxis) of MOs, with PDGF was potentiated by nearly 60%. This effect was specific for PDGF and was a maximal response at 3pmol bound. MO haptotaxis was inhibited by three different maximal at TSP concentrations 10-fold lower than those required for SMC migration mAbs recognizing distinct sites on the COOH terminus. As with chemotaxis, the 140- to TSP alone. Since mAb C6.7 completely abolished chemotaxis in response to the kD fragment, but not the HBD, contained the haptotactic activity. The CSVT- combination of PDGF and TSP, it appeared that cell surface-associated TSP might be containing synthetic peptide also promoted MO haptotaxis. In contrast to chemotaxis, critical in SMC chemotaxis to PDGF. We confirmed this by disrupting TSP-SMC neither PT nor CT inhibited TSP-mediated haptotaxis, suggesting a different signal interactions using mAb C6.7 or by pre-incubating SMC with cycloheximide before transduction pathway. mAbs against GPIV, pl P3, or ot integrins did not affect MO exposing them to PDGF. In both cases, SMC chemotaxis in response to PDGF was chemotaxis or haptotaxis, thus ruling out the involvement ofthese receptors. These significandly inhibited. These studies indicate that TSP is a potent autocrine motility results indicate that TSP could play an important role in directing MO migration. factor for SMC and is essential in PDGF-dependent SMC migration.

1061 1062 EFFECT OF GEL ELASTICITY ON FIBROBLASTS EMBEDDING IN IN VITRO AND IN VIVO EFFECTS OF EXTRACELLULAR COLLAGEN GELS. ((J. Kamimura and K. Mochitate)) Environ. Health MATRIX AND GROWTH FACTORS ON VESSEL FORMATION. Sci. Div., Natl. Inst. Environ. Studies, Tsukuba, Ibaraki 305, Japan. ((N. Fournier & C. J. Doillon)). St-Frangois d'Assise's Hospital Fibroblasts in collagen matrix contract collagen gel and generate and Laval University, 10 del'Espinay, Ou6bec, (Que), Canada, tension In the tissue and the tension affects fibroblasts on cell shape, G L 3L5. growth and biosynthetic activities. Because tension developed by gel contraction depends on tissue elasticity, it Is interesting to compare Specific extracellular matrix (ECM) and growth factors (GFs) behavior of fibroblasts embedded In soft and stiff gels. Two kinds of appear to control endothelial cell behavior and angiogenesis. type collagen, with and without telopeptide region (I-A and I-P, Among ECM, fibrin clot is the primary matrix for angiogenesis respectively), were examined: gels were 10-fold in e I-A stiffer lstkity after injury. In the present study, than I-P gels. Fibroblasts in I-A gels retained the initial population GFs and various during 1-day culture and then started to proliferate. Flbroblasts In I-P concentrations oftibronectin (FN) or hyaluronic acid (HA) were gels, however, lost -40% of the initial population at1st day of culture. combined to a fibrin matrix model. In vitro, the migration of Without protease treatments to digest the tissues, 14C-thymidine- capillary endothelial cells and the size of vessels were prelabeled fibroblasts also exhibited the similar decrease in radio- quantitatively determined as a function of molecule activity asin cell population. Protein synthesis was, in contrast, -60% compositions within fibrin. Low concentrations of HA or FN lowerin I-A gels than in I-P gels. As type lIl and V collagen ae known significantly increased migration distance of cells and to interact with type collagen, we also examined cell behavior in decreased the size of microvessels independently on the mixture gels of type I andIl-V collagen. By adding collagen type I-A, presence of GFs. In contrast, high concentrations of HA or FN 11, III or V to type I-P at the ratio of 0.2:1.0 (w/w),gelelasticity Increased induced large microvessels, particularly in the presence of and the initial population of fibroblasts was almost retained. Only type IV collagen tested decreased gel elasticity and the population GFs. In vivo, the adsorption of fibrin matrices containing HA, decreased. Protein synthesis in the mixture gels of type I and type I-A, FN and/or GFs to a polyester yarn modified angiogenesis that 11, III or V collagen also reduced to the same level as that In only I-A occurs within implants. In addition to their biological properties, gels. These results suggest that population and protein synthesis of HA, FN and GFs could modify the structure of fibrin gel fibroblasts In collagen gels are regulated by tissue elasticity resulted matrices and consequently the migration of cells and the from association of matrixcomponents and limited proteolysis. fibrinolysis rate during vessel formation in vitro and in vivo. Monday. Basement Membranes: Synthesis and Biochemistry (1063-1068) 183a

1063 1064 CHARACrERIZATION OF THE MOUSE ENTACTIN GENE. IN VITRO SYNTHESIS AND ASSEMBLY OF DERMAL-EPIDERMAL JUNCTION ((M. E. Durkin, J. Reing, and A. E. Chung)) Departnment of IN DUAL SPECIES CULTURES ((T. Nishiyama, A.M. McDonough, R. Bruns, Biological Sciences, University of Pittsburgh, Pittsburgh, PA and R.E. Burgeson.)) MGH/Harvard Cutaneous Biology Research Center, 15260 Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Entactin, also known as nidogen, is a 150 kDa basement In vitro models of skin development utilizing keratinocytes grown upon membrane protein that binds to laninin, type IV collagen, cells, fibroblast-contracted collagen gels have been known to accumulate and calcium ions. Sequence analysis shows that it is a multidomain basement membrane (BM) components at keratinocyte-gel junction. protein that contains motifs found in other proteins, including 6 However, contribution of keratinocytes and fibroblasts to BM synthesis, EGF-like cysteine-rich repeats. We have screened mouse genomic structure and assembly is little known. To elucidate contribution of the DNA libraries in order to detennine the strucal organization of cells to the BM formation, dual cultures of fetal bovine keratinocytes the gene and to locate potential transcriptional regulatory regions. (BK) and human fibroblasts (HF) were analyzed by species specific Genomic clones that contain over 80kb of the gene have been antibodies. Kalinin (KN), type IV collagen (IV), laminin (LN), type VII isolated, with some gaps remaining. At the 5' end, approx. 30kb collagen (VII), and type XIl collagen (XII) were synthesized by BK alone of DNA have been obtained, including the first exon, which and were localized to the BK-gel (without HF) junction. IV, LN, VII, and encodes the 5' leader of the mRNA, the signal peptide, and the first Xl were also synthesized by HF in presence of BK and were localized to the 47 amino acids of the mature polypeptide. The first exon and the BK/HF-gel junction. In contrast, KN was produced only by the surrounding DNA are G+C rich and appear to comprise a CpG keratinocytes. The similar results were observed in culture for 8, 16 and island. The region upstream of the transcription start site has 30 days. The results strongly support that both cells contribute the BM putative TATA, CAAT, and GC boxes and displays promoter synthesis and deposition in the junction. In ultrastructural evaluation, BM activity in transient trnsfection assays. The second exon is structures, such as continuous lamina densa, lamina lucida, separated fron the first by a very large intron that extends grater hemidesmosomes, and anchoring fibrils, were observed in the BK/HF-gel than 18 kb. Exons 2-5 are clustered within 9kb and encode the junction of 16 and 30 day-culture. The junction in 8 day-culture was first globular domain (GI), the hydrophilic, protease-sensitive poorly organized, regardless of the localization of BM components as hinge region, and the first EGF repeat. The EGFrepeats appear to above, but some hemidesmosomal structures were observed even at this be encoded by distinct exons, consistent with the idea that entactin stage. The results indicate that the BM assembly of the components occurs and other multidomain proteins evolved by "exon-shuffling." much later than synthesis and hemidesmosomal structures are formed at first stage of BM assembly. Formation of hemidesmosomal zone may be a clue of BM assembly.

1065 1066 THE BINDING OF FIBRONECTIN TO ENTACTIN IS MEDIATED THROUGH A NEW MOLECULE COVALENTLY ASSOCIATED WITH KALININ THE 29 kDa AMINO TERMINAL PEPTIDE FRAGMENT OF FIBRONECTIN AND ((M-F. Champliaudl, T. Nishiyamal, A.M. McDonoughl, D.R. Keene* THE G2 DOMAIN OF ENTACTIN. Hsieh#, C. Wu and A. E. Chung#)) ((J.-C. and R.E. Burgesonl.)) I Cutaneous Biology Research Center, #Departmnent of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, Massachusetts General Hospital and Harvard Medical School, Boston, MA and Mayo Clinic, Scottsdale ,AZ 85254. and * Shriners Hospital for Crippled Children, Portland, Oregon. Previous work has shown that fibronectin and entactin, an ubiquitous basement membrane glycoprotein, colocalize in the extracellular matrix of the The kalinin molecule has been described as a molecule specifically embryonal involved in the anchoring filaments of carcinoma-derived 4CQ cell. In addition, the direct association of the two the hemidesmosomes. Recently, Marinkovich et al. (1992) demonstrated the association of molecules has been demonstrated by solid phase binding assays. However, the have the kalinin to K-laminin is composed 1 domains through which the two molecules interact have not previously been molecule K-laminin. of three chains: B and B2 chains of laminin and an A'-chain supposed to identified. In order to obtain further information, glutathione-S-tranferase (GST) be closely related to the A- fusion proteins containing different domains of entactin have been obtained by chain of kalinin. During the steps of the purification of the complex Kalinin -K-laminin from human amnion, we have been able to a subcloning entactin cDNA sequences in the pGEX3X vector and expressing the isolate new complex involving and a new molecule. This recombinant sequences in E. coli JM109. The expressed GST-entactin fusion kalinin molecule would be composed of the B2 chain of laminin, B Is chain of S-laminin described by proteins, GST-G1, GST-G2, GST-E and GST-G3, were extrcted with 6 M urea in Hunter et (1989) and A-chain of kalinin. 100 mM Tris-HCI, pH 9.0. After renaturation the solubilized fusion proteins were al. the Further evidence of this complex obtained by purified with a glutathione affinity column. By using a solid phase binding assay, have been immunofluorescence using the Bls antibody kindly provided by Dr Sanes and the kalinin antibody. In amnion, it was shown that both human fibronectin and the 29 kDa amino 1251-labeled the two antigens co-localised in the stromal-epithelial junction. In terminal fragment of bovine fibronectin bound specifically to the immobilized GST- addition, the observations of the purified fraction after rotary shadowing show a G2 fusion protein but not to GST-Gl, GST-E, and GST-G3. Half saturation for complex molecule with two long arms and two short arms. We propose the binding of the fibronectin 29 kDa to the immnobilized GST-G2 fusion fragment name of KS-laminin for this new molecule associated with kalinin. Cell protein was obtained at a concentration of approximately 5nM. It is suggested that adhesion assays have been performed with this complex. the strong association between GST-G2, which contains the second globular Keratynocytes attach to the complex and are specifically detached by the BM-165 domain of entactin, and the amino terminal-29 kDa of fibronectin may be involved of kalinin A-chain. in the assembly of certain types of extracellular matrices. Furthermore, the antibody, specific the Mechanism of the adhesion and determination of the epitope of the antibody are in progress. association may modulate the biological properties of the two molecules. [Supported by grants CA 21246 and GM 25690 from the National Institutes of Healthl.

1067 1068 BASEMENT MEMBRANE GLYCOCONJUGATES. ((M.W. Smithson, FIBRILLAR LAYER ASSOCIATED WITH THE BASEMENT MEMBRANE OF THE and N.A. Kurpakus)) Department of Anatomy and Cell INNER ENAMEL EPITHELIUM OF THE DEVELOPING TOOTH IS A LAMINA Biology, Wayne State University School of Medicine, DENSA-LIKE STRUCTURE RICH IN BASOTUBULES. ((T. Sawada. and S. Detroit, MI 48201. Inoue)) Department of Oral Pathology, Tokyo Dental College, Chiba City, Japan, and Department of Anatomy and Cell Biology, The ocular surface of the eye is composed of McGill University, Montreal, Quebec, Canada. H3A 2B2 corneal, limbal and conjunctival epithelium. We hypothesize that the molecular composition of the Basement membrane-associated fibrillar layer of the inner basement membrane that underlies the ocular surf ace enamel epithelium in early stages of odontogenesis was examined. epithelium is tissue specific, and have therefore Tooth germs of the monkey were observed at high magnification initiated an analysis of the glycoconjugate in the electron microscope. A "fibril" within the fibrillar composition of this structure. Frozen sections of layer appeared as a 10 nm-wide pentagonal frame with a lumen, bovine ocular surface epithelium prepared from in cross section, and as a set of three parallel lines, in "young" eyes were analyzed with a panel of FITC- longitudinal section. Such a structure is typical of conjugated lectins. Several lectins, which recognize "basotubules", which are believed to be microfibrils localized galactose residues, were localized to the corneal within the basement membrane. Each of the spaces between these and limbal basement membrane. In contrast, these structures was occupied by a network of 4 nm-thick, irregular lectins did not label the conjunctival basement anastomosing strands resembling "cords" as found in the basement membrane. These lectins were soybean agglutinin membrane. For immunohistochemical studies, tissues from various (SBA), wistaria floribunda agglutinin (WFA), and species including the monkey, rat and dog were subjected to vicia villosa agglutinin (WA). Lectin staining was pre-embedding immunoperoxidase staining. Cord-like strands in specific, as it was abolished by competitive binding the network immunostained positively for laminin, type IV to the sugar galactose. In "adult" cow eyes, we were collagen and heparan sulfate proteoglycan (HSPG). The structures unable to detect binding of SBA, WFA and WA to which immunostained for HSPG were identified as 5 nm-wide either corneal or conjunctival basement membrane. "double tracks", the form known to be taken by HSPG in basement Based on lectin analyses, we speculate that there membranes. In summary, the so-called fibrillar layer which is may be age-related changes in the glycoconjugate associated with the dental basement membrane is composed of a composition of the corneal basement membrane. cord network as observed in basement membranes, and is also rich in basotubules. 184a Basement Membranes: Synthesis and Biochemistry (1069-1074). Monday

1069 1070 GLYCATION OF LAMININ: A MODEL FOR ALTERATION OF BASEMENT INTERACTION OF TRANSFORMING GROWTH FACTOR P WMT THE MEMBRANE ARCHITECTURE IN AGING AND DIABETES. (P.D. BASEMENT MEMBRANE PROTEOGLYCAN PERLECAN. ((M. R. Austia and Yurchenco and Y.-S. Cheng)). Department of Pathology. Robert Wood J. R. Ccschman)) Deprtent ofCdl gy, Unisity ofAlabam at Johnson Medical School. Piscataway, NJ. 08854. Birminghan, VH201C, Birmigam, AL 35294.

Non-enzymatic glycosylation (glycation) of basemant membranes has Extrceular matrix moleus can seqeae growth faScto which may the affdct previously been proposed as a mechanism for the developmant of struc- prolifer migration md diffentation evente mtiu. Baweat membane tural and functional changes in aging and diabetic microvascular disease. matrix is dqepskd by the amne endodemal celline PFHR-9, and when cell-frve We evaluated the effects of glucose and ribose on basement membrane matices were prepared by l dent mehods, the pratio reduced laminin in vitro. In molar excess (0.75M), labeled glucose and ribose of fieshly PFHR-9 cdls. Bloking antibodies aginst were incorporated into laminin with apparent pseudo-first order kinetics TGF- I overcame this effect, did heparin (but nat chondrotin sulft) eabnent, and similar binding capacities (175 and 184 mol sugar/ mol laminin, re- orh eparinanHand I, (but not nd tina ABC) treatments of matic befor spectively). Glycation resulted in the progressive inhibition of laminin soding frsh cells. Furte, the mink hmg eptial cell line (MvlLu), known for itAs polymerization (short arm function) and heparin binding followed by the growth supprson by TGF-P, was also growth inhibited by cell-free PFHR-9 formation of specific fluorescence (335-340 nm excitation/ 420 nm matics. This too, was ovecm by bepari (but not chondtin sul;fae) or emission maxima) and inter-chain, intramolecular covalent crosslinks in heprinae andamM (but nat chdroinas ABC) treames ofmatrices. Th TGP- the long arm. Noticeable molecular shape changes were not observed by is probably secreted in a lAent form, since no bioativity was detctable in PPHR-9 electron microscopy (Pt/C replicas). Previously we have shown that a cture growth ofundiubed PFHR-9 menlayes was not a fraction of laminin exists in collagen IV network-bound pool p e ely suppresed, and wesem blottig of matrix gave a 1 lOkD polypide, When [Yurchenco, Cheng & Colognato, 1992, J. Cell Biol. 117: 11191. cnist with latat TGF-P. The alklie hydr is method usod for maix this pool (EHS tumor) was glycated, most laminin was released into so- prparatin may actethe TGF-P. Perecn abundant m PFHR-9 matrix, and lution with a portion remaining bound to the collagen-IV network in a TGFlI was shown to bind puified preca thrugh the beparan slfae chais. non-polymerized state. The data are compatible with a model in which Therefore, TGF-P may bind bawSnt membrane p cans, wher it can become exposure of basement membrane laminin to elevated glucose results in activad to inf cell bebavior. work was spwr by NIH gmnts the gradual inactivation and displacement of laminin into solution and AR36457, AR 39741, and American Het Grantin-Aid 9100636. JRC is an network in a more porous with the residual laminin bound to the collagen Estblished Inetgator ofMme American Hrt Association. state. (Supported by NIH grant R01 -DK41 500).

1071 1072 SKIN FIBROBLASTS ARE MAJOR PRODUCERS OF LAMININ AND NIDOCGE. SUBSTRATE ANDASCORBATE EFFECTS ON BASEMENT MEMBRANE (R. Fleischmajer, M. Bruns, J.S. Perlish, D. MacDonald, R. PRODUCTION AND ORGANIZATION BY HUMAN VASCULAR ENDOTHEUAL Tiripl, T-C. Pan and M-L Chu). Dlepartment of Dermatology, CELLS. ((A.C. Hospelhom, C.M. Milbury, W.M. Abbott, and R.W. Orkin)) Moutic Siniai Medical Center, New York, NY 10029; Department of Laboratory of Vascular Cell Biology, Surgery Research Unit, Mass. General Biochemistry and tMolecular Biology, Jefferson Medical Hospital, Harvard Medical School, Charlestown, MA 02129. College, Philadelphia, PA 19107, Max Planck Institut fur Biochemie, Martinsried, Germany. Endothelial cell (EC)-extracellular matrix interactions may influence the outcome of vascular tissue remodeling by altering EC function. We have Laminin and nidogen are components of basal mmina. Although examined the effects of exogenous substrates on the endogenous pro- their structure is well known, their tissue origin is not duction, deposition, and organization of basement membrane collagen clear. This problem was approach with an "in vitro" system (COL IV), the matrix upon which EC reside In vivo. EC were isolated from where fibroblasts are grown into a nylon mesh (dermal model adult human saphenous veins and from human umbilical veins and cultured or DM) and then recombined with keratinocytes (Advanced on wet films of gelatin (GEL) or fibronectin (FN) In the presence or ab- Tissue Sciences). This results in the formation of an sence of ascorbate (ASC). Cultures were examined at 2-3 day intervals epidermis, basal lamina, anchoring zone, dermis and a lower from preconfluence to postconfluence. COL IV production and organiza- monolayer of fibroblasts (Keratinocyte-dermal model or R-DM). tion by saphenous vein EC were both substrate- and ascorbate- The epidermis was then separated from the dermis with dependent. By day 9, COL IV levels were 10-fold higher in cultures of thermolysin. Immunofluorescence microscopy ard Western blots saphenous vein EC on FN +ASC and 2-fold higher on FN -ASC than In those were performed with antibodies against laminin and nidogen. on GEL +ASC. Immunofluorescence studies revealed that the organization Northern blots and "in sitv" -ybri.dization were performed was a dynamic, time-dependent process. Under all culture conditions, the with specific cDNA's against nidogen (1Kb segment) and the B2 COL IV matrix originally appeared as a meshwork, which was subsequently chain of laminin (2.2Kb). Imaunofluorescence and Western reorganized into thick cords, except on GEL -ASC, where It disappeared. blotting revealed only niciogen in D-M while the K-DM revealee The reorganization occurred at a faster rate by EC grown on GEL +ASC both, nidogen and laminin. Northern blots and "in situ" than in cultures grown on FN. In contrast, COL IV deposition and organi- hybridization studies revealed that nidogen was an exclusive zation by human umbilical vein EC were ascorbate- but not substrate- product of fibroblasts. Most of the laminin was also produced dependent. The dramatic changes in COL IV organization observed In by fibroblasts although small amotunts may be produced by the saphenous vein EC cultures did not occur In umbilical vein EC cultures. epidermis. This study suggests that fibroblasts are major These differences highlight the heterogeneity of human endothelial cells producers of laminin and nidogen but the epidermis may and indicate potential functional differences In the biological responses of regulate their polymerization into the basal lamina. these cells.

1073 1074 PDGF STIMULATES SECRETION OF BASEMENT MEMBRANE LAffNIN EXPRESSION IN THE DEVELOPING AVIAN EYE. ((W.- PROTEINS BY MUSCLE CELLS. ((D.E. Albrecht, M.J. Spencer, and J.G. H. Tang and J. J. O'Rear)) Department of Molecular Genetics and Tidball)) Dept. of Physiological Science, Univ. of California, Los Angeles. Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ. 08854 Laminin, collagentype IV, and fibronectin are basement membrane proteins that can influence muscle growth and differentiation. In this study, we investigate whether growth factors can influence secretion of basement To investigate the role of basement membrane laminin and its genetic membrane proteins by stimulating L6rat myoblasts with platelet derived variants in eye development, we have used cross-species nucleic acid growth factor (PDGF) in yin. L6 myoblasts stimulated with 20 ng/ml hybridization to isolate from an adult chicken eye library partial PDGF-BB for 24 h showed an increase in laminin and collagen type IV recombinant cDNA clones for chicken laminin A, and merosin. accumulation in the culture media by 115% and 188% respectively, relative Ble, B2e, DNA sequence analysis of these a new to unstimulated cultures. Less than 35% of this increase was attributable to candidate cDNA clones identified increased myoblast proliferation during the assay. Stimulation with higher B1 chain variant (J. Biol. Chem. 267: 20555-20557) and confirmed the concentrations of PDGF (50 ng or 100 ng/ml) produced no further increase identity of the other clones. The new variant is unusual in having a high in laminin or collagen type IV secretion. No significant increase in (80%) amino acid sequence identity with laminin B1 in the N-terminal fibronectin concentration was produced by PDGF stimulation at any portion of domain VI yet differs in a sequence conserved between the tested. Supernatants collected from of L6 myoblasts A, concentration cultures B1, and B2 chains from Drosophila to man. Further characterization of pulse stimulated for 10 min with 20ng/nil of PDGF-BB showed at the end the primary structure of the new of 24 h an incrase in laminin, by 218% relative to controls, and collagen variant indicates that it diverges in the type IV, by 66%. Fibronectin, collagen type IV and laminin in extracts of C-terminal portion of domain VI and in domain V. These cDNAs will myoblasts stimulated for 24 h with 20ng/mI PDGF showed no change in be useful probes to analyze the temporal and spatial pattern of concentration relative to extracts of unstimulated myoblasts. Thus, PDGF expression of laminin and its variants in the developing avian eye. applied to L6 myoblasts for either 10 min pulse orchronically for up to 24 h resulted in elevated secretion of laminin and collagentype IV, but no change in fibronectin secretion. In view of the absence of basement membranes on myoblasts, we propose that PDGF-stimulated increase in laminin and collagentype IV secretion relates to ancillary functions of these proteins in myogenic tissue, such as providing chemotactic stimuli for other myogenic cells and neurons. (Supported by NIH AR-40343.) Monday. Basement Membranes: Synthesis and Biochemistry (1075-1079) 185a

1075 1076 THYROID CELLS CULTURED IN MONOLAYERS OR FOLLICLES SYNTHESIZE IDENTIFICATION OF BAMIN AS THE ACUTE PHASE, SERUM PROTEIN A LAMININ VARIANT. ((H. Feracci and F. Andre)) U270 INSERM, HEMOPEXIN AND THE IMPLICATIONS FOR INDUCED POLYCYSTIC Fac. de Medecine, Marseille, France; Dept. of Cell Biology and Anatomy, Johns Hopkins Medical School, Baltimore, MD 21205 KIDNEY DISEASE. ((D.H. Rohrbach and R.R. Koepsel)) Center for Biotechnology & Bioengineering and the Department of Microbiology/ Basement membranes are thin specialized extracellular matrices Biochemistry, University of Pittsburgh, Pittsburgh, PA 15219 known to be involved in cell attachment and proliferation as well as modulation of differentiation of epithelial cells. As In previous work, the authors extracted a glycoprotein from the basement in many other tissues, thyroid epithelial cells rest upon a membrane extracellular matrix of the EHS tumor and demonstrated that this basement membrane and we have detected A and M heavy chains of laminin (LM) and merosin respectively, both surrounding folli- glycoprotein was significantly reduced in amount in mice with induced cles. This structure has never been detected with these cells polycystic kidney disease. This glycoprotein was tentatively named bamin. in primary cultures. We have investigated the ability of This report presents data showing that bamin is the serum protein, hemopexin. freshly isolated porcine thyroid cells, seeded on plastic and Mouse bamin and rat hemopexin show specific antibody cross reactivity, have organized in monolayers or in follicular structures, to synthe- similar peptide migration patterns on SDS-PAGE, both bind heme, and have size and deposit LM. These cells are polarized in both organi- zations but express different levels of thyroid functions. We nearly identical amino acid compositions. Partial sequence of mouse bamin have shown de novo synthesis of a trimeric protein (210, 220, shows nucleotide and amino acid identity to rat hemopexin of 92 and 87%, 380 kDa) immunoprecipitated with an antiserum against EHS-LM, respectively. Identification of bamin as hemopexin may be critical to our in both of our culture systems. This molecule is an isoform of understanding of the pathogenesis of induced polycystic kidney disease. the classical EHS-LM, with differences in the heavy chain. Hemopexin is an acute phase protein that functions to remove excess heme Immunofluorescence labeling indicate that the protein is secre- from ted in a polarized fashion and is, at least in part, deposited the serum during periods of stress. Loss of bamin/hemopexin could cause on the basolateral side of the cells in both organizations. The periodic increases in serum levels of heme that could damage the renal filtration identification of the heavy chain synthesized in vitro is cur- unit, the basement membrane. Increases in heme could also damage the rently being investigated. This molecule is turned over rapid- epithelial cells lining the renal tubules, eventually resulting in the formation of ly in vitro but is degraded differently in the two different structural organizations. cysts. Preliminary data indicate that loss of hemopexin and the resultant high heme levels may, indeed, be part of the pathogenesis of polycystic kidney disease.

1077 1078 OVEREXPRESSION OF NM23 IN HUMAN BREAST CARCINOMA CELLS cDNA CLONING OF HUMAN S-LAMININ REVEALS INDUCES ACINUS-LIKE SPHERE FORMATION, BASEMENT THAT IT IS EXPRESSED BY CARCINOMA CELI-S. MEMBRANE DEPOSITION AND GROWTH ARREST. ((A.R. Howlettl, O.W. ((U.M.Wewer and R. Albrechtsen)) University Institute of Petersen2, P.S. Steeg3 & M.J. Bisselll)) 1Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720 2Panum Institute, University of Pathological Anatomy, Copenhagen, Denmark Copenhagen, Denmark 3Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892 (Spon. by J. Campisi) A partial cDNA clone encoding human S-laminin mRNA was isolated and characterized. This 2 kb fragment encompasses We previously developed an assay system for the study of human breast domain La,. Comparison with rat S-laminin shows 92.7 % cancer where normal breast luminal cells and breast tumor cells recapitulate similarity. Comparison with human BI shows 55.1 % similarity. many aspects of their in vivo patterns of growth and differentiation when The motor-neuron adhesion-promoting tripeptide LRE (Hunter placed within a matrix of reconstituted basement membrane. We used this assay to investigate the function(s) of nm23, a putative metastasis suppressor et al Cel 59:905, 1989) was not present in the predicted human gene. Human metastatic MDA-MB435 breast carcinoma cells transfected amino acid sequence. Instead LQE and LRG were found at two with nm23-H1 revert to an almost normal phenotype within reconstituted of the corresponding locations. We have examined the basement membrane in that they form small breast acinus-like spheres from expression of S-laminin in a series of human carcinoma cell single cells, deposit a type IV collagen- and laminin- containing endogenous lines in culture using this cDNA as a probe together with basement membrane and show a significant reduction in growth. Occasional monoclonal antibodies generously donated by Dr JR Sanes (St spheres form a central lumen although they are otherwise unpolarized by the criteria of sialomucin deposition at the apical membrane and lack of cadherin Louis, MO). We found that all cell lines tested expressed S- expression. Parent MDA-MB435 cells and control transfectants form large laminin mRNA at level comparable to BI laminin mRNA. S- disorganized colonies that fail to deposit an endogenous basement membrane laminin polypeptide was also detected by Western blotting. and grow continuously. These data suggest that nm23 may play a role in Moreover, it appeared by double immunofluorescence staining basement membrane formation. Furthermore, the deposition of an that the same tumor cell produced both S and BI chains. Thus membrane correlates with arrest and formation endogenous basement growth we conclude that cultured human carcinoma cells produce both of more normal, luminal epithelial-like, three dimensional structures within reconstituted basement membrane. Bi and S laminin.

1079 SPECIFIC STIMULATION OF BASAL LAMINA HEPARAN SULFATE PROTEOGLYCAN IN MOUSE UTERINE EPITHELIUM BY MATRIGEL MAY IN PART BE DUE TO SEQUESTERED TRANSFORMING GROWTH FACTOR-Bl. ((John E. Morris, Georgeen Gaza, and Sandra W. Potter)) Department of Zoology, Oregon State University, Corvallis, OR 97331-2914.

The basal lamina of differentiated epithelium normally turns over only slowly unless stimulated by tissue repair and growth. A large heparan sulfate proteoglycan (HSPG) extracted from mouse uterine epithelium was recognized by a specific monoclonal antibody (from Koji Kimata) to the basal lamina HSPG, perlecan. Compared with other HSPG, perlecan showed specifically enhanced ["Slsulfate labeling in extracts from cultures on the basal lamina preparation Matrigel. Partial stimulation was obtained with a serum-free medium extract of Matrigel, which fractionated on Sephadex G- 50 into two components; a major one > 30 kDa and the other 15-25 kDa. The specific stimulation was mimicked by the addition of 10 ng/ml of TGF- B1, but there was no specific stimulation by basic fibrobUast growth factor (which, however, did show general proteoglycan stimulation), epidermal growth factor, insulin-like growth factor-1, or interleukin-1. TGF-B1 was identified as a 12.5 kDa monomer in thiol-reduced Matrigel (4-25 ng/ml) and in Matrigel extracts (1-3 ng/ml) by polyclonal antibodies (from R&D Systems) on transblots following SDS-polyacrylamide gel electrophoresis. Failure of excess amounts of these antibodies to inhibit Matrigel-stimulated basal lamina HSPG synthesis indicates that they were unable to interact with TGF-B1 bound to Matrigel and/or that TGF-B1 is only one component of Matrigel that is important in stimulating basal lamina HSPG synthesis in culture. The fact of only partial stimulation by Matrigel extract or TGF-I1 supports that latter conclusion. We suggest that in vivo TGF-B1 is bound to macromolecular components of mouse uterine epithelial basal lamina, where it may be sequestered until microenvironmental changes make it available to promote basal lamina HSPG synthesis. (NIH grant HD-1 9530) 186a Extracellular Matrix and Integrins in Morphogenesis I (1080-1085). Monday

1080 1081 FORMATION OF EXTENSIVE CANALICULAR NETWORKS BY RAT HEPATOCYTES SPONDLYLOMETAPHYSEAL DYSPLASIA N MICE WrrH A DOMINANT NEGATIVE GROWN IN A COLLAGEN SANDWICH CONFIGURATION. ((E. L. LeCluyse, K. MUTATION N TYPE X QO"QEN_ ((O. Jacenko, P. LuValle, B. R. Olsen)) Cell L. Audus, and J. H. Hochman)) INTERx Research/Merck Research Biology, Harvard Medical Boston, MA 02115 0. Laboratories and the Departments of Biochemistry and School, (Spon. by Jacenko.) Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047. The vertebrate skeleton forms primarily by endochondral ossification (EO) when bony trabeculae and marrow replace cartilage. During this Rat primary hepatocytes were cultured between two layers of process, hypertrophic chondrocytes express a unique matrix molecule, collagen gel (Vitrogen5) and evaluated for the acquisition and collagen X. To define its function In EO, transgenic mice with a dominant maintenance of structural and functional cell polarity. Cultures negative phenotype for collagen X were generated (DNX, Inc.). Constructs of sandwiched hepatocytes formed an extensive and contiguous network of bile canaliculi (BC) within 24-48 hr. BC formation in included the chick tl(X) cDNA with inframe deletions, inserted between the hepatocyte cultures grown under conventional single gel conditions chick c (X) promoter and upstream regulatory elements, and CAT. was much less extensive and more variable in rate. The Localization of the transgene product to hypertrophic cartilage confirmed that canalicular network exhibited distinct morphological changes with the chick 5' regulatory elements were functional in mice. 14 transgenic time which were reflective of the ultrastructural and functional lines, each with an Independent transgene integration site, showed similar maturation of the BC. Iimmunostaining of 'sandwiched cultures for apical membrane markers (aminopeptidase N. dipeptidyl peptidase skeletal defects. Of the genotypically positive pups, 20% become hunch- IV) demonstrated localization predominately at the canalicul ar backed -day 17 after birth, with pareses of hind limbs, and die. Additional structures. Rhodamine-phalloidin staining of filamentous actin phenotypic defects Include dwarfism in some homozygotes. Histology of iong showed a marked rearrangement of the intracellular microfilaments bones and vertebrae of genotypically positive mice showed compression of to the cell periphery that coincided with the development of the hypertrophic growth plate cartilage, reduced hypertrophy, an altered BC. Acquisition of normal BC function and integrity was observed hypertrophic chondrocyte pericellular matrix, and reduced bony trabeculae, within 3-4 days postoverlay as indicated by the concentration and retention of carboxyflourescein and fluorescein-conjugated but no mineralization abnormalities. Mice with the hunch-back phenotype taurocholate within the canalicular network. These results showed, in addition to the above defects, leukocyte deficiency In the marrow, demonstrate that hepatocytes grown in a sandwich configuration are a reduced spleen and thymus, and lymphopenia. Growth plate abnormalities capable of complete re-establishment of normal cell polarity. suggest that collagen X may have a structural role during skeletogenesis, and This system may serve as a more reliable and representative model that mutations In COLIOAI are responsible for certain human in which to study the physiology of hepatic function as well as chondrodysplaslas. (Supported by NIH AR36818 & 36820, NRSA AR08030 the morphogenesis of polarized membrane domains in vitro. & 07922, Arthritis Investigator Award, and NOI-HD-0-2911.)

1082 1083 THE GLOBULAR REGION OF LAMININ B CHAIN(S) IS CRITICAL CLONING AND CHARACTERIZATION OF THE SEA URCHIN FOR EPITHELIAL CELL POLARIZATION IN THE DEVELOPING LAMININ Bi CHAIN. ((E.M. Laxson and J.D. Hardin)) Cell and LUNG. ((L. Schuger, *A. Skubitz and K. Gilbride)). Dept. of Pathology, Molecular Biology Program, University ofWisconsin-Madison, Boston University School of Medicine, Boston, MA 02218 and *Dept of Madison, WI 53706. Lab. Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455. Taminin, a large glycoprotein complex, is a major component of Laminin has been shown to promote lung branching morphogenesis basement membranes and is involved in cell adhesion, cell (Schuger et al, Dev. Biol. 146: 531-41, 1991). This study is part of an migration, cell differentiation, and morphogenesis. In order to effort to determine the mechanisms underlying this effect. AL-5 A study the role oflaminin in a convenient embryonic context, we monoclonal antibody against the globular region of the B chain(s) (Skubitz have cloned and are currently sequencing the laminin Bi chain et al, J. Biol. Chem. 263: 4861-68, 1988) was used to interfere with from sea urchin embryos. Using the Polymerase Chain Reaction morphogenic cell-cell and cell-matrix interactions occurring in an (PCR) and primers specific for region VI ofthe B1 chain, we organotypic culture system. In this system, mixed cell populations from amplified a 90 bp fimgment from a cDNA library constructed from embryonic lungs rearrange in a pattern resembling the tissue of origin. Cells late gastrula stage Lytechinus variegatus embryos. The fragment is were isolated fronm the lung and preincubated with various concentrations of 75% homologous to the corresponding segment in Drosophila AL-5, antibodies to other laminin sites, and control immunoglobulin. The melanogaster laminin Bi. Screening the cDNA library with the cells were then induced to rearrange organotypically. AL-5 did not affect PCR of cell aggregation and sorting into epithelial and mesenchymal compartments. probe yielded four positive clones. Partal sequence analysis However, the antibody prevented epithelial cell polarization and subsequent the cDNA clones reveals homology with the Drosophila, human, lumen formation. The other treatments did not affect organotypic pattern and mouse B1 chains. Initial sequence analysis ofthe largest clone formation. The lack of cell polarization was not the result of alterations in (4.2 kB) reveals that it spans the cell-binding domain ofthe BI epithelial or mesenchymal cell attachment, as demonstrated in adhesion chain (which in vertebrates contains the peptide sequence YIGSR) assays. Immunohistochemical studies using antibodies to major basement which will permit analysis oflaminin's role in sea urchin membrane (BM) constituents revealed absence of BM formation in the gastriuation. We plan to use Northern blot analyis to determine cultures exposed to AL-5. These findings suggested that the globular region the temporal expression oflamninin B1 in sea urchins and to build of laminin B chain(s) may be critical in the process of BM assembly and cell bacterial fusion constructs for antibody production. Complete polarization. sequencing ofthe obtained clones and the entire B1 chain coding region are currently underway.

1084 1085 ANTIBODIES TO LAMININ AND INTEGRINS INHIBIT IDENTIFICATION OF A NOVEL CELL ADHESION SITE IN THE ES FOLDING OF THE AVIAN OTIC PRIMORDIUM. ((R. Visconti, REGION OF LAMININ WITH A ROLE IN LUNG ALVEOLAR S.R. Hilfer)) Department of Biology, Temple University, Phila, PA FORMATION. ((M.L. Matter and G.W. Laurie)) Department of 19122 Anatomy and Cell Biology, University of Virginia, Charlottesville, VA 22908. During the early development of the otic vesicle, a longitudinal fold is formed along the lateral wall of the neural tube. The basal surface of the primordium is closely associated with the lateral wall An in vitro model system was used to investigate the role of laminin of the neural tube between stages 11 and 14. The basal lamina in lung alveolar morphogenesis. Freshly isolated rat type 11 glycoprotein laminin and putative laminin receptors of the integrin alveolar cells form alveoli-like structures when cultured on family have been immunolocalized throughout the anlage during basement membrane. To identify the alveolar promoting site(s) on early organ formation. Monoclonal antibodies raised against laminin laminin we initially divided laminin into its proteolytic fragments E8 and CSAT antibodies have been used to investigate the role of their and P1. Alveolar formation was inhibited by the carboxy-terminal respecdve antigens in the attachment of the otic primordium to the laminin E8 fragment but not the P1 fragment. Alveolar promoting neural tube. Stage 10 embryos, injected with either antibody, were was cultured overnigt and frozen sectioned or prepared for scanning activity then localized, through the use of antibodies and electron microscopy. Results show that attachment to the neural synthetic peptides, to a 20 amino acid sequence (SN-pep; tube and subsequent invagination were interrupted by both #2179-2198) within the first loop of the laminin A chain G domain. antibodies. Monoclonals raised against laminmn were labeled with Activity was further defined by trypsin digestion of the peptide to the BODIPY for in vivo studies of the roles of laminin binding and sequence SINNNR. This highly conserved site promoted divalent turnover in otic development. Fluorescence was viewed in intact cation dependent adhesion of both type 11 and HT1080 cells. Cell embryos in culture at low light intensity using a Quantex 200 ICCD adhesion to E8 was completely inhibited by an equimolar amount camera. Frozen sectioned or embryos prepared for TEM were of SN-pep. In immunological studies, antibodies to the SN-pep processed to intensify the staining of the sites of antibody binding. sequence stained fetal alveolar basement membranes, which This procedure will be used to localize the binding of antibodies raised against the laminin receptors of the integrin family. This demonstrates the presence and steric accessibility of this site. SN- research is supported by a grant from the Deafness Research pep is a novel adhesion site within the E8 region of laminin which Foundation. plays a prominent role in lung alveolar formation. Supported by CTR grant # 2933R2. Monday. Extracellular Matrix and Integrins in Morphogenesis I (1086-1091) 187a

1086 1087 THE IN SITU LOCALIZATION OF TENASCIN AND THROMBOSPONDIN 2 HYALURONAN IS INVERSELY CORRELATED WITH THE mRNAs IN THE DEVELOPING CHICK ((R.P. Tucker and P.M. Poss)) EXPRESSION OF CD44 IN THE DERMAL CONDENSATION OF Department of Neurobiology and Anatomy, Bowman Gray School of THE EMBRYONIC HAIR FOLLICLE ((C.B. Underhill)) Department of Medicine, Wake Forest University, Winston-Salem, NC 27157. Anatomy and Cell Biology, Georgetown Medical Center, 3900 Reservoir Road, Washington DC 20007 Tenascin-C, the founding member of the tenascin family of glycoproteins, is concentrated in the extracellular matrix (ECM) of the In the present study, we examined the distribution of CD44 (the in and and hyaluronan receptor) and hyaluronan in the skin of embryonic and mature developing brain and spinal cord, early cartilage bone, lining mice. During embryonic development, CD44 was prominently expressed the migratory pathways of neural crest cells.- Several splice variants of by the condensed mesenchymal cells involved in the formation of the hair tenascin-C have been identified. To determine if tenascin-C variants follicles, but was absent from the surrounding interstitial cells. The cells of potentially have different functions, we have used in situ hybridization the dermal condensation expressed CD44 throughout the development of the and RT-PCR to localize splice variant transcripts. mRNAs encoding hair follicle, however, once the hair follicle reached maturity, the tenascin-C with the variable FN type III repeats "A" and "Bo are found in a mesenchymal cells of the dermal papilla no longer expressed this molecule. subset of proliferating and migrating glia in the chick spinal cord, as well In contrast to the above, the distribution of hyaluronan was reversed from as in epithelial glia. In contrast, tenascin-C transcripts without repeats that of CD44. Hyaluronan was wide spread throughout the embryonic "A" and "B" are found in cartilage perichondrium. PCR products dermis, but was conspicuously absent from the regions of the dermal corresponding to the 3 most abundant splice variants can be amplified condensation. This arrangement persisted through the development of the hair follicle, however, in the mature hair follicle, hyaluronan reappeared in from poly(A) RNA isolated from trunk neural crest cells by RT-PCR. This the dermal in that neural crest cells themselves are the papilla. Thus, the embryonic dermis, the expression of CD44 provides further evidence and hyaluronan were complementary to each other. However, in the adult source of the tenascin-C found lining their migratory pathways. Uke the skin, only minor changes were detected in the levels of CD44 and tenascins, thrombospondins (TSPs) are a family of glycoproteins found in hyaluronan associated with the cells of the dermal condensation during the the CNS and developing connective tissues. We have constructed a hair cycle. When organ cultures of embryonic mouse skin were treated DNA probe to one member of this family, TSP2. TSP2 transcripts are with Streptomyces hyaluronidase, the interstitial mesenchymal cells became not detectable in the developing CNS by in situ hybridization or by compacted, indicating that the removal of hyaluronan leads to the northern blotting. Instead, TSP2 mRNAs are found in cartilage condensation of these cells. The results of this study are consistent with the perichondrium, epimysium and endothelial cells. Thus, TSP2 is probably hypothesis that the expression of CD44 by the inductive mesenchymal cells playing a role in connective tissue, but not brain, morphogenesis. allows them to degrade hyaluronan in a localized region, leading to formation and maintenance of the dermal condensation.

1088 1089 INTERACTION OF NEURAL CREST CELLS WITH CHONDROITIN MONOCLONAL ANTIBODIES AGAINST LINK PROTEIN ALTER FEATHER GERM SULFATE PROTEOGLYCAN IN VITRO AND IN VIVO ((Z. Pettway, DEVELOPMENT IN CULTURED EMBRYONIC SKIN. ((Franqois Binette, Behnamn R.Perris, and M. Bronner-Fraser)) Developmental Biology Center, U.C. Kahoussi and Paul F. Goetinck)) Cutaneous Biology Research Center, Massachusetts Irvine, CA 92717 General Hospital, Harvard Medical School, Charlestown, MA 02129. Link Protein (LP) is an imporant component of the extracellular matrix of cartilage Chondroitin sulfate proteoglycans are abundant around the notochord where it forms a ternary complex with hyaluronic acid and the large aggregating in avian embryos. Cartilage-type proteoglycans that contain chondroitin, proteoglycan, aggrecan. The interaction between aggrecan and hyaluronic acid is keratan and dermatan chains are typically found in the perinotochordal significantly stronger in the presence of LP, indicating a stabilizing role of LP in the region. Previous results have shown that chondroitinase treatment of complex. We recently reported on the presence of LP mRNA and its translation product in notochords in culture and prior to implantation abolish the notochords a wide variety of non-carlinous tissues (Binette et al. MoL Biol. Cell. 3: 224a;1992). ability to inhibit neural crest cell migration. Such results suggest a role Furthermore, we have shown in vitro that LP can enhance and stabilize the interaction of for chondroitin sulfate proteoglycans (CSPG) in inhibiting neural crest proteoglycans isolated from skin to hyaluronic acid (Binette et al., submitted 1993). In cell migration. We have used two assay systems to further test the the present study, we performed an immunohistochemical analysis of the localization of inhibitory effects of chondroitin sulfate proteoglycans on neural crest LP during skin development, We found that LP is detectable in the mesodermal cell migration: 1) direct comparison of neural crest behavior in vit-o on condensation of the early feather buds. In 10-12 day embryos, LP staining is observed in adjacent lanes of CSPG and fibronectin; and 2) implantation onto neural the entire dermal component of the feather. In an effort to address the in vivo function of LP in non-carilaginous tissues, we studied the effect of monoclonal antibodies on the crest of microcarrier filters coated with migratory pathways itn vivo development of feather germs in organ cultures of embryonic skins. When seven day old CSPG or fibronectin. In the in vitro assay, neural crest cells were added embryonic skins were grown in the presence of a monoclonal antibody directed against LP to neighboring lanes of fibronectin adjacent to various forms of purified (3H8/C3), developmentally retarded feather buds developed only in the central rows. The CSPG. The results demonstrate that neural crest cells preferentially development of feather buds of the lateral rows was inhibited. A monoclonal antibody migrate on the fibronectin-coated lanes, whereas they are not observed which recognizes Carfilage Matrix Protein (lGl/Al0), used as a control antibody, had no on the CSPG lanes. In the in vivo assay, neural crest cells were observed effect. These results suggest that LP is involved early in feather development and provide on the surface of fibronectin-coated microcarriers and appeared to the first evidence for the biological significance of the presence of LP in a non- migrate toward them even when they were outside the normal migratory cardlaginous tissue. (Supported by NIH grants HD 22016 and HD 22050) pathways; in contrast, neural crest cells only were observed adjacent to CSPG-coated microcarriers when they were placed directly into the migratory pathway. These int vit-o and in vivo results support the idea that fibronectin and CSPG differentially affect the behavior of neural crest cells. (Supported by USPHS HD-15527)

1090 1091 MICROINJECTION OF ANTI-NCAM INHIBITS INVAGINATION REDISTRIBUTION OF A BASEMENT MEMBRANE PROTEIN DURING OF AVIAN MORPHOGENESIS OF MUSCLE AND HYPODERMIS. ((M.C. Hresko and R.H. INNER EAR. ((S.R. Hilfer, J.W. Brown, and E.D. Beck.)) Waterston)) Dept of Genetics, Washington University Sch. of Med., St. Louis, MO. Department ofBiology, Temple University, Philadelphia, PA 19122 061 10.(Spon. by P. Hoppe.)

During inner ear development, the otic placode folds to form a box-like Linkla between contractile filaments and the extracellular matrix are important for force otic vesicle. Folding is inhibited by microinjection ofeither antibody to tnmi e, and specific linking proteins may help specify the position of attachment stuctures In C. ekgaas body wail muscle, contactile filments are anchored trally to lhe laminin or enzymes which degrade chondroitin sulfate or hyaluronan. membrne by dense bodies Zdiac analog) and M-lines, which in turn are anchored to the Agents affecting the cytoskeleton have no effect. Thus, folding appears cticle (outer covering of the worm) by a specialized bsoment menbrame ad hemid.oatsnes to be driven by changes in extracellular matrix under the lateral region of of the hypodnmis (cellular syacitium tht covers de worm and seces the cuticle). A the placode (paraxial mesoderm) and between otic epithelium and neural potenil molecular link, beginning at thc muscle cell, extending through the basement membe and so the l' has been identified immu lgilyt. tube. Expansion ofextracellular matrix may exert a pushing force on the The MH46 tigen, a basment membrane protem prst between muscle cells and the otic epithelum. For a pushing force to be effective, the epithelium must cutice may assod wih nmule stuctus The aIen is ynt zed be anchored to underlying cells and also remain an intact sheet. Cell by doal ad ventrl hypodeemis but becomes coextensive with the assembling muscle, into rows of does that nm to the axis of the worm, an adhesion molecules serve this function. As a first step we are testing the obliquely long suggesting with the obliquely striaed body wall muscle. Later in development, associton of role ofneural cell adhesion molecule (NCAM), which is expressed early the MH46 angen with becomes evident. when the staining patem detected in development ofthe otic primordium. Antibody to NCAM which is similar to that with antibodies to prtin or with MH46 in adults. blocks function (gift ofDr. C-m Chuong and from Developmental Thes results ae consste with at least two rols for the MH46 antgen. 1) Analysis of muscledefective munts suggests the posiion of strucul elments of body wal muscle is Studies Hybridoma Bank), was injected with a picospritzer underneath dictatd by membrane proximal events3. Since the MH46 atign is a basment membne the ectoderm of intact stage 10 chick embryos. Photographs of living protein asoied with mwscle during its assembly, it may be involved in positioning the embryos and scanning electron micrographs show a range from slightly stuctural elemen of the muscle. 2) The MH46 antigen may serve to strengthen links distorted primordia to drastically inhibited folding on the injected side. between the lattice and hemidesmosomes. The MH46 antigen asociates with heIdeImoso_m during the latw sges of embryogenesis through adulthood, but may also The actual site ofantibody blockage is being investigated to correlate the mainta interacto with muscle, which are evident ealier in developMent. Tis would degree of inhibition with possible NCAM function. Supported by a provide for coely cWpd,piodic Hlnks between muscl andhe m grant from the Deafness Research Foundation. We we currently attempting to coe, and map the p encoding the MH46 tigen. This knowledge will elp in designing sceens for mu ns affecting the MH46 gen product that will be used to study th in Wvo role of the prtein. 1,2) rancis and Waton. 1985,1991. 3) Hresko, Wl_ms, and Werston 1993, submitted. 188a Extracellular Matrix and Integrins in Morphogenesis I (1092-1095). Monday

1092 1093 TRANSGENIC EXPRESSION OF STROMELYSIN FROM THE WAP INTEGRIN DISTRIBUTION IN EARLY MACAQUE PLACENTAS ((C.S.T. PROMOTER ALTERS BRANCHING MORPHOGENESIS DURING Pow', T.N. Blankenship2, B.F. King2, and A.G. Hendrickx')) 'California Regional MAMMARY GLAND DEVELOPMENT AND RESULTS IN PRECOCIOUS Primate Research Center and 2Dept. of Cell Biology and Human Anatomy, Univ. EXPRESSION OF MILK GENES. ((C.J. Sympson*+, C.M. Alexander%, J.R. of California, Davis, CA, 95616. Chin*, Z. Werb', and M.J. Bissell+)) 'Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143, and Placental growth involves cell proliferation, production of extracellular matrix +Life Sciences Division, Lawrence Berkeley Laboratory, CA 94720. (ECM) and interactions of trophoblast with ECM. Laminin, fibronectin, and type are in the distal cell columns and The extracellular matrix (ECM) is an important regulator of the IV collagen abundantly produced trophoblastic differentiated phenotype in the mouse mammary epithelium. ECM- shell of the placenta. ECM interactions with cells are thought to be mediated by degrading metalloproteinases (MMPs) and their inhibitors have been integrins. Therefore, we studied the distribution of integrin subunits in developing implicated in tissue morphogenesis and ECM remodeling. MMPs are macaque (lacaca fascicularis) placentas (27-34 days of gestation) using regulated during normal development of the gland in vivo: MMP expression immunoperoxidase methods. The basal surfaces of villous cytotrophoblasts maximal is minimal during lactation, when the gland is fully functional, and a6 and subunits. In the cell which are during involution, when the gland loses mammary-specific function. To expressed 04 cytotrophoblastic columns, examine the role of MMPs in ECM remodeling and their importance in areas of cell proliferation, differentiation, and migration, expression of a6 and 04 mammary gland morphogenesis, we have generated transgenic mice that subunits decreased from proximal to distal regions. In contrast, al and aS density inappropriately express autoactive forms of rat stromelysin (SL) in the increased along the proximal-distal axis of the columns. p1 was weakly expressed mammary gland under the control of the mammary gland-specific whey in the columns. Trophoblast in the shell expressed al, a5, and.01 but was acidic protein (WAP) promoter. Unexpectedly, there was expression of the negative for a6 and p4. Expression of integrin subunits may alveolar provide trophoblast transgene in the virgin gland, giving rise to precocious development. cells with information differentiation based on local environmental The virgin transgenic glands morphologically resembled glands from normal controlling mid-pregnant mice. These alveoli that developed in the virgin transgenic cues from the ECM. Also, fetal trophoblast cells interact with maternal ECM as glands were capable of synthesizing beta-casein at a similar level as a normal they undergo limited invasion of the endometrium. In macaques the border mid-pregnant gland, compared to scarcely detectable levels in nontransgenic between the shell and ECM-rich endometrial stroma is even. The distinct virgin controls. These data suggest that MMPs are involved in branching distribution of each of these receptor subunits suggests close regulation of integrin morphogenesis and that the modulation of cell-ECM interactions in vivo and ECM expression in placental tissues and may play a role in the cessation of affect epithelial cell organization and function. (Supported by USDOE- into the endometrium. NIH RR00169 OHER #DE-AC03-76-SF00098, DE-AC03-76-SF01012, NIH CA57621 and trophoblast migration Supported by grants ES07 106). and HD24491.

1094 1095 INTEGRIN-MEDIATED ADHESION OF MOUSE PERI-IMPLANTATION 01 INfEGRINS MEDIATE THE THREE-DIMENSIONAL ORGANIZATION OF BLASTOCYSTS IS ACTIVATED BY IMMOBILIZED LIGAND. ((J. F. Schultz HEART CELIS IN AN IN VITRO MODEL OF CARDIC MORIHOGENESIS. and D. R. Armant)), C. S. Mott Centerfor Human Growth and Development, Dept. of ((T.Bacro, T.Borg, LTewraclo d W.C wer)). De mst of Bilog OB/GYN, Wayne State University School of Medicine, Detrot, Ml 48201 sad AwAemy. Uiversky of South Caoln Scool of Medicn. Cohaia, SC 29206. Previous studies have suggested the involvement of Integrins in trophoblast Cardic sa of ell prolifeaon, interactions wih endometrial extracellular matrix during blastocyst implantation. In the morphogenesis complex dIferentIton, tion ad sort at tim present study, we used fluorescert microspheres coated wth the central ceN binding specifk peiods in fragment of fibronectin (FN-120) to localize and characterize fibronectin (FN) binding emnbryon lTe that direct the 3-dienaonal or tin of activity on the surface of mouse blastocysts. Fluorescence was quantified on the beart cel and ECM are poorly undestood; bowever, cel-mat Interactions embryo suriace using a computer-based Image analysis system. Initially, we found bave been ggested to be und tal i this proces Te prest study wan - in that the FN binding activity was correlated with blastocyst antachment (24 48h undertaken to Investi the role of specdfe componets of the ECM and their - 96h in FN culture), rather than trophoblast cell adhesion and outgrowth (72 culture). integrin receps In bet cell aggation sort. Neonat rat both FN- binding activity of blastocysts cultured for 48h was competitively inhibited by cardic and fibroblts were bolted and maintained 6 days 120 and heparan sulfate. In addition, microsphere binding was significantly (4-5 days) myocytes In rotation culture either In medium with 10 % newbor serum 5% fetal bovine decreased after heparinase was used to remove heparan sulfate from the surface of the embryo. Thus, embryos cultured in the absence of extracellular matrix up to 96h sum (SM) or In serum free medium (SFM. The ressdts sbow that 1) exhibited no integrin-mediated FN binding hctivity. To determine whether fibroblasts and myocytes are Iitiaylly co-mingle In the aggregates, 2) by day 2 immobilized FN-120 could induce FN binding activity, 72h blastocysts were cultured sortn is apparent with the fibroblats seen predomat In the outer portions on dishes precoated with FN-120 for 3h, treated with heparinase, and incubated with of aggregates and myocytes In the inner portions we cultured with SM and 3) The FN-1 20 coated microspheres for 30 minutes at 4°C. ligand-activated blastocysts cells fal to sort In SFM. he addition of TGF-B and bFGF but not PDGF to did not to bound FN-1 20 at the abembryonic pole, however, 48h blastocysts respond Immunocytochenial stainn of fibronectin this treatment. Free FN-120, but not heparan suiate or other FN domains, SFM Iduced sorting. l1minin (LN), competitively inhibited FN binding activity. Binding activity was also inhibited by the () and 01 integrin (51) was randomly distibuted ia 2 day aggregtes. By 6 synthetic peptide, GRGDSP, but not GRADSP. Removal of divalent cations using days, the staining of LN and FN wa asocated with ECM and 51 with cel EDTA inhibited binding, which was restored by the addtion of 1mM MgCI2, 5mM surface of central myocytes and peripheral fibroblats Sorting wa completly MnC12 or 10mM CaC12. Antibodies raised against either the P1 or p3 integrin subunH bocked by pre-Incbation of heart cels with antibodie agalnt FN and 51, but blocked FN binding activity, while non-immune sera did not. These results indicate not by LN, collagen I, N-edmrin or N-CAM IgG's. The 3-dimeiona activated that the integrin-mediated FN binding activity of mouse blastocysts was by organiation appears to be dependent on cell-m i Intio mdated by p1 exposure The of to this to immobilized ligand. capacity embryos express ligand- Integrins Furte studies wil focus on specife a chain(s) which mediate the both and with the activated binding correlated temporally spatially developmental mechanism In this from grts HL37669 and HL42249). program regulating trophoblast cell adhesion. Supported by NIH grant HD 25795. sorting model.(Support Visual Systems (1096-1097)

1096 1097 RETROGRADE TRANSPORT OF NEUROTROPHINS IN ADULT RAT EXPRESSION AND REGULATIONOF ACTIVIN A ANDFOLLISTATIN INHUMAN RETINAL GANGLION CELLS. ((J. Beer, L. McKerracher, C. Essagian, RETINAL PIGMENT EPT LIAL CEILS (hRPE). ((CIL Harris WI. Robet GJ. A. Aguayo)) Ctr. for Research in Neuroscience, McGill Univ.and jaffe)) Dqea et of O y, Duke Univerdty, Durha, NC 27710 Montreal General Hospital Res. Inst., Montreal, Quebec H3G 1A4. lo e To investigate the populations of retinal ganglion cells (RGCs) that Activin A is a icmolele compisIng 2 BA submis It is a mbser of the respond to brain-derived neurotrophic factor (BDNF) we have exam- TOM fanily of gtwth far and was origiay idenffied as agou lda lvedpoeen dhat ined in vivo the retrograde transport of BDNF. BDNF was iodinated stimulae FSH. Subhanuently, it whfound to have cell-pecificeffoct an cel (sp. act. 2-8 X 1O' cpm/pg) and 400 ng was injected into the pr1llfsatitt, dlfetundtio, wound rpair and n l Poistan is amonomeric tectum (n = 6 animals), or into optic nerves crushed 9 mm from the g dnwhich bid acvim tbeteb Ihi-idh avnms c..lb actiites. lbem eye (n = 10 animals). The biological activity of BDNF was confirmed is littl infomato on the expNin of thes protins by ocular tissue Te goal of ths by the appearance of label in dorsal root ganglion (DRG) cells after study wa toIcbacterne theexpesn of activin A ad follst hinn hRPE and D demine BDNF was injected into transected sciatic nerves. The animals were n fixed by perfusion, and cryostat sections of the retina or DRG were whetereNxpresion isregulated by thecys on IL-1Bad T1FL To e mRNA processed for emulsion autoradiography. Labelling of RGC neurons expreation_, o RNA wan extedhn cullrdhRPE, mve raiwd tocDNAand was observed after the application of BDNF to the tectum, with a amplfied with PCR usn prime specific foractvin BA chain or fosatin. BA chai and density that suggests most RGCs transport BDNF. This finding folstatin mRNA were both eprssedconstntvely in 10/10 cel lines tested. Mhe level of indicates that BDNF may provide trophic support for most RGCs in BA chainexprssionwas inrasd in adosedepenatmanmerby both IL-lB and TGC adult mammals, as suggested by the temporary rescue of virtually folstain mRNA levels were aftr expr to The all axotomized Slmilaly, eiacod thes agma. RGCs by BDNF (Mansour-Raboey et al. (1992) addiion of protIn to in Molec. Biol. Cell 3:333a). Application of BDNF to the crushed optic tbe synthss hbitorc IL-1Breulted squnction and nerves was not an effective route of application, perhaps because of the BA chain butnotfoOistatm. Levels of IL-lB TGFB e incead in ocular g the uptake of BDNF differs in sciatic and optic nerves. We are infammatoy and woundheab diseases, which in ta may lead to ineasedproduction of examining if RGCs are also capable of retrogradely transporting the wadvin A. The cooatledregulao of activin A and follista sugests that foistltn can other members of the neurotophin family of growth factors. serv to atamust actfvi-mediaeddfexs Monday. Visual Systems (1098-1103) 189a

1098 1099 CENTRIN-LOCALIZATION IN THE PHOTORECEPTOR CELLS OF CLONING AND CHARACTERIZATION OF THREE PHOTOTRANSDUCTION MAMMALlAN RETINAE ((U. Wolfrum)) Dept Biochemistry & Molecular GENES FROM XENOPUS LAEVIS. ((S. BATNI, J.Q. WANG, S. Biology, Mayo Foundation, Rochester, MN 55905; Inst. of Zool. I, Univ. SUNDARESWARAN, B.E KNOX)) Department of Biochemistry and Molecular Karlsruhe, Germany. (Spon. by A.T. Baron.) biology, SUNY-HSC, Syracuse, NY 13210. ( Spon. by K.MeirL ) To better understand the mechanisms involved in the control of in the Mammalian photoreceptor cells are polarized ciliary sensory organs. Their transcription vertebrate retina, we have chosen to study the regulation of the photoreceptor cell outer are modified segments specialized, light-sensitive organelles, resembling transducin subunit and arrestin Our cilia. Their membrane forms flattened membrane specific genes: opsin, alpha inXenopus. approach amplified ciliary stacks, has been to clone and characterize their cDNAs and corresponding genomic clones are termed disks, which packed with the visual pigment The connecting chium containing upstream sequences for promoter analysis. The cDNAs for all three genes serves to join the outer with the inner segments and the cell bodies containing were isolated from an adult retinal cDNA library, and showed a relatively high degree of all metabolic active organelks. Therefore, the connecting cilium appears to homology with their bovine counterparts. Nucleotide and deduced amino acid identities play an important roe in photoreceptor organization. However, little is known of: 77% and 73% for opsin, 80% and 93% for transducin and 68% and 66 % for arrestin, about the molecular composition of this ciliary structure. The present study respectively, have been found. Protein encoded by the opsin cDNA, produced in transient shows that the cytoskeletal calcium binding protein centrin is present in the transfections of COSI cells and purified by antibody affinity procedure, exhibited an photoreceptor cells of the mammalian retina. Centrin is a 20 kDa EF-hand absorbance maximum of 502 nm, which was close to that observed for the abundant (red) phospho-protein that forms fine contractile filaments in green algae. Using rod opsin inXenopus retina. Sequence analysis of the opsin gene indicates the presence monoclonal antibodies centrin is detected in western-blots of purified fractions of five exons interrupted by4 introns. The position ofexon-intron borders in theXenopus of the photoreceptor cells. In addition, anti-centrin immunofluorescence occurs opsin gene was conserved with those of opsins from other vertebrates. Upstream in these cells. Moreover, immunoelectron microscopy reveals that centrin is sequence comparison ofXenopus opsin with other vertebrate opsins reveals homology localized in the cilium and the lattice of the basal bodies of within 350 nudeotides from the transcription start site, suggesting potential promoter mainly connecting function. The characterization of transducin and arrestin genes and their upstream the cells. These indicate that photoreceptor findings Ca2+-triggered motile sequences is in progress. To study promoter function, 5.5kb and 4.4kb of upstream processes based on centrin may occur also in the mammalian retina. sequence from opsin and transducin alpha subunit genes respectively, were cloned into a Preliminary experiments indicate that the orientation of the basal bodies luciferase reporter vector.Promoter activity is being asayed following lipofectin- relative to each other is, indeed, dependent on the intracellular Ca2+- mediated transient transfections of stagedXenopus embryos. Cis-acting elements will be concentration. Additionally, centrin-based motile processes may be involved in identified by deletion analysis. TMis work was supported by grants from the March of intracellular transport between the inner and outer segment of the Dimes and National Institutes of Health. photoreceptor cells as well as in the adaptive retinomotor movements. Supported by the DFG (Wo 548/1-1)

1100 1101 CONE TRANSDUCIN (Tca) PROMOTER TRANSGENE EXPRESSION IN RETINAL DOCOSAHEXAENOATETRAFFICKING AND PREFERENTIALUPTAKE lTHE MOUSE RETINA. ((E. Bogenmann and S. Cooper)) Departments of BY PHOTORECEPTORS. ((W.C. Gordon, E.B. Rodriguez de Turco, and N.G. Pediatrics and Microbiology, Childrens Hospital Los Angeles, Los Angeles, Bazan)) LSU Eye Center, New Orleans, LA 70112. CA 90027; and University of Southern California School of Medicine, Los CA 90033. Angeles, Docosahexaenoic acid (22:6ta3 or DHA) is synthesized from its essential precursor acid and then removed from The mouse retina is composed of a number of highly specialized neurons a-linolenic (18:3Xa3) by liver, rapidly including a classes of photoreceptors, namely rods and cones. Mouse cones are systemic circulation by retinal pigment epithelial cells (RPE). DHA is released further subdivided into blue (short wave) and red/green (medium wave) to the interphotoreceptor matrix, to be taken up by inner segments of cones. Only about 2-4% of all photoreceptors are cone cells and this has photoreceptors, where it is esterified into phospholipids for subsequent hampered the analysis of cone cell development. We describe the membrane assembly. DHA has been localized autoradio-graphically in frogs characteristics of mouse strains expressing the temperature sensitive (ts) large to outer segment discs, RPE and cone oil droplets, and synaptic vesicles of in T-antigen (LTag) gene driven by the promoter of the Tca gene. A 1.8-kb vivo and in vitro preparations. Purpose: This study was designed to establish promoter fragment was used to generate 4 transgenic lines. Three lines so far a distribution pattern and time-course for [3H]DHA trafficking from the time it analyzed show a spatially uniform expression pattern of the nuclear enters retinal cells. Mefhods: Injections of [3H]DHA were followed by retinal oncoprotein throughout the retina only in cells apposed the pigmented fixation after 1 h to 34 d. Some retinas were isolated and incubated in [3H]DHA epithelium. No ts-Tag-induced retinal degeneration occurs as has been for up to 6 h. Conventional light and EM autoradiography were used. Resulfts: described for the wild type oncogene. Expressing cells have been identified Retinal distribution varied. RPE cells labeled first, followed by photoreceptor as cone cells based on double immunofluorescence, using peanut agglutinin lectin and the anti-large Tag antibody. Postnatal expression is first detected inner segments. Oil droplets of both cells types also labeled. By 6 h, at P1 by antibody staining, and at P0 by RT-PCR, coinciding with the photoreceptor synaptic terminals were labeled. Outer segments became expression of the endogenous Tca gene. Embryonic expression remains to be diffusely labeled by 1-6 h, but newly assembled, densely labeled discs were determined. Developing retinas from blue opsin-lac Z and Tca-tsTag present at 1 d. Generally, only rods became well labeled; cones labeled expressing mice enables further insights into cone cell commitment and diffusely, except for oil droplets. Only rod synaptic terminals became heavily differentiation, since the lac Z transgene is also expressed in cone bipolar labeled. After shedding, [3H]DHA was removed from phagosomes and directed cells. Mouse strains carrying the diphtheria toxin (DTA) gene under the back to receptor inner segments. Relatively few changes occurred in the rest control of the Tca-promoter are currently used to further study the role of of the retina. Conclusions: DHA is differentially taken up by photoreceptor cones in outer nuclear layer differentiation. cells. Once esterified into phospholipids, it is directed either into new discs or into synaptic vesicles. Rods accumulate the majority of this unique fatty acid. Support: EY04428

1102 1103 ACTIVE DE NOVO SYNTHESIS OF DHA-PHOSPHATIDYUNOSITOL IN THE IRON INDUCES METALLOTHIONEIN (MI) IN CULTURED RETINA. ((E.B. Rodriguez de Turco, W.C. Gordon, and N.G. Bazan))LSU Eye HUMAN RETINAL PIGMENT EPITHELIAL (RPE) CELLS. Center and Neuroscience Center, LSUMC, New Orleans, LA 70112. ((P.D. Oliver, D.J. Tate Jr., and D.A. Newsome)) Touro Infirmary and Tulane University School of Medicine, New Orleans, LA 70115 Docosahexaenoic acid (DHA, 22:6n-3), the main polyunsaturated acyl group of from disc membranes and terminals of phospholipids (PL) synaptic Free iron in the presence of H202 has the potential to form hydroxyl photoreceptors, is required for their normal function. DHA-PL display the radicals via Haber-Weiss reactions. In the absence of both ferric highest de novo synthesis (Aveldano and Bazan, Neurochem. 1:381, 1980). serum, and ferrous iron caused an increase in radiolabeled zinc Incubation of frog (Rana pipiens) retinas with [3H]DHA for 1-8 h, showed an uptake by early peak in DHA-PA labeling coupled to and active labeling of DHA-PI, cultured human RPE cells. To investigate whether the action of iron was followed by phosphatidylethanolamine (PE) and phosphafidylcholine (PC). At due to an induction of MT, we used a cadmium/heme assay to measure these early times, most of the labeling was detected autoradiographically in the MT and found that ferric iron (3 FuM, 18 hr) caused a 2.5-fold increase in myold and ellipsoid regions of photoreceptors. Retinas were also labeled with MT levels (n = 9, p = 0.0001). This induction was inhibited by the rH]DHA in the presence of propranolol, an amphiphilic cationic drug that addition of two compounds capable of scavenging free radicals, DMSO blocks the de novo synthesis of major PL (i.e. PE, PC) at the level of (1 %) or n-acetyl cysteine (NAC, 20 mM). Induction of MT also phosphatidate phosphohydrolase. Labeling of PC and PE was decreased by occurred after a 2 hr incubation with ferric iron (3 AM), which was 80%, indicating that most DHA is introduced In these PL by de novo synthesis inhibited by adding NAC or cycloheximide (1 mM). Iron (3 AM, 4 hr) rather than turnover. Moreover, propranolol induced higher [3H-DHA also caused an increase in binding of NF-kB to its DNA binding motif, incorporation into PA, Pi, and polyphosphoinositides. Because this pathway an effect that was inhibited by addition of NAC during the stimulation. is decreased In retinas of with rod-cone selectively dogs progressive To determine if free iron is involved in the induction of MT by H202 or deTurco et its degeneration (Rodriguez al., IOVS, 1993) up-regulation by drugs phagocytosis of photoreceptor outer segments, we preincubated the cells such as propranolol may contribute to the restoration of DHA-PA and DHA-PI with an iron desferrioxamine 2 which in the retina and Improve function. chelator, (100 FM, hr), abrogated pools photoreceptors Supported by MT induction either condition. Our results demonstrate that NEI/NIH grant EY04428. by intracellular free iron is capable of eliciting a cellular stress response involving induction of specific gene expression in RPE cells. The induction may be mediated by the interaction of iron with H202 and subsequent generation of hydroxyl radicals. 190a Visual Systems (1104-1105). Monday

1104 1105 OXIDATIVE STRESS INDUCES CATALASE, METALLOTHONEIN IMMUNOCHEMICAL LOCALIZATION OF PHOSPHOGLUCOMUTASE AND NF-kB IN HUMAN RETINAL PIGMENT EPITHELIUM (RPE). IN SKELETAL MUSCLE AND NEURAL RETINA. ((M. Pd*, A. ((D.J. Tate, Jr., D.A. Newsome, and P.D. Oliver)) Touro Infirmary and Bayardot, M. Martonet, M. Ellismant, P. Boundis*, and RB. Marchase)) Tulane University School of Medicine, New Orleans, LA 70115 *Dept. of Ccli Biology, Univ. of Alabma at Bim Birminghm, AL 35294-0005 and tDept. of Neurosciences, Univ. of California, San Diego, Reactive oxygen intermediates have been implicated in the aging process LaJolka, CA 92093-0608. and degenerative diseases of the eye, including retinopathy of prematurity, cataractogenesis and macular degeneration. Age-related decreases in both Phosphogucmutas (POM) i a cytoplanic enyme that intercoverts Gc- catalase and metallothionein (MT), cellular antioxidants, have been shown I-P and Glc-P and ha recendy been shown to be glycosylted. Biochemical to occur in RPE cells. The purpose of the present study was to investigate fiac on of rat skeletal musce by our laboratoy ad others (Lee et al., J. whether RPE cells respond to the oxidative stress of phagocytosis of Chem 267:21080 1992) has shown PGM is partially associat with photoreceptor outer segments or to addition of exogenous H202. Our study th rcoplasmic reticulum (SR). To examine this in more detaiL indirect shows that an 18 hour exposure to either stress induces the following three immunofluorescence was performed on cryostat sections of rat skeetal muscle cell parameters: 1) catalase enzyme activity (n =7, pO0.05), 2) MT (n=4, with monoclonal antibodies shown to be specific for PGM by Westen blot p50.05; measured by a cadmium/heme binding assay) and 3) the DNA analys and immunopiptation. PGM was found to be enriched in the binding of the nuclear regulatory factor NF-kB (gel retardation assay). junctional SR and colocalized with CaATPase, calsequestrin, and the ryanodine Activity/mg protein Control H20, Phagocytosis receptor. In addition, the neural retina was dissected from albino rabbits, Catalase (Units) 56 8 103 9 105 18 prepared for indirect immunofluorescence and examined. Preliminary anayses MT (.tg) 4.9 0.9 12.2 0.23 10 0.24 demonted that PGM was especialy concenaed in MUller cells in the the Addition of n-acetyl cysteine, a free radical scavenger, abrogated myelinated band region of the refina Subcellar loalization sesd that, or addition of H202. Our results inductive effect caused by phagocytosis as in nwscle, PGM was not bcalizing as a ubiquitous cytoplasic protein. demonstrate that the RPE cell response to phagocytosis is indistinguishable from the response observed following the addition of exogenous H202. Generation of H202 during phagocytosis may act as an intracellular signal in RPE that leads to increased levels of key antioxidant enzymes and other proteins important for protecting the cells from oxidative damage. Gene Structure II (1106-1109)

1106 1107 DNA DAMAGE AND REPAIR IN HUMAN TELOMERES. ((P.A. INCREASED FREQUENCY OF APOLIPOPROTEIN B SIGNAL PEPTIDE Kruk, N. Rampino, and V.A. Bohr)) Laboratory of sp24/24 IN PATIENTS WITH CORONARY ARTERY DISEASE. ((J. Molecular Genetics, National Institute on Aging, H.Wu, M.S.Wen, S.C.Kao, S.K.Lo, M.S.Chern, D.Wu)) National Institutes of Health, Baltimore, MD 21224. Department of Microbiology and Immunology, Chang-Gung Medical College; Cardiology Section, Department of Telomeric regions of chromosomes play an essential Internal Medicine, Chang-Gung Memorial Hospital, Kwei- role in the maintenance of chromosomal stability. San, Tao-Yuan, 333 TAIWAN. Decreases in telomeric length are seen with increasing donor age, increasing passage in culture, The apolipoprotein B signal peptide (sp) polymorphism and tumorigenesis. We have established a technique was characterized by polymerase chain reaction in 371 to study DNA damage formation and its repair in individuals of which 313 were samples from the normal telomeres in human cell lines. Cultured cells are population and 58 were from coronary artery disease irradiated with ultraviolet (UV) light and the DNA (CAD) patients. The frequency distributions in normal is isolated, digested with Hinfl, and treated with group were 0.81 sp27 (sp with 27 amino acids) and 0.19 T4 endonuclease V which cleaves DNA at pyrimidine sp24 (sp with 24 amino acids); in CAD group, they were dimer sites. The samples are separated by alkaline 0.77 sp27, 0.23 sp24. The genotype distributions were gel electrophoresis, transferred, and hybridized 0.64 sp27/27, 0.33 sp27/24 and 0.03 sp24/24 in normal group; 0.64 0.26 sp27/24 and 0.10 sp24/24 in with end labeled (TTAGGG) 4 or (AATCCC) 4. Auto- sp27/27, radiograms are scanned and the data are analyzed for CAD group. The frequency of sp24/24 in the CAD group the frequency of pyrimidine dimers and their removal was significantly higher (p-O.012) than that in the by DNA repair. In the present study we have compared normal group. Other studies have shown that the DNA damage in telomeres with that in an endogenous frequency of sp24/24 is higher in hyperlipidemic group gene. The time course of repair in the telomeres is than in normolipidemic group. The marker is probably comparable to DNA repair in non-transcribed regions in linkage disequilibrium with soma other atherogenic of the genome. This study also compared telomeric genes. Our study also showed that statistically repair capacitites with age as well as strand significant differences existed between Chineses and specificity of repair. Irrepairable telomeric damage Caucasians in the apoB signal peptide allele and sp27/ may contribute to chromosomal instability leading to 27 and sp27/24 genotype distributions. cellular senescence or, possibly, transformation.

1108 1109 EVOLUlTON OF MUSCLE ACTIN GENES: A Pf-YLOGENETIC STRUCTURE AND COMPLETE SEQUENCE OF THE CHICKEN ANALYSIS. ((ME White and B.I. Crother)) Departmnt of Biological SMOOTH MUSCLE y-ACTIN GENE. ((A.M. Kovacs, and W.E. Sciences, Southeastem Louisiana University, Hammond, LA 70402. Zimmer)) Department of Structural and Cellular Biology, University of South Alabama School of Medicine, Mobile, AL 36688. Actin proteins are encoded by a multigene family in all animals studied to date. In the vertebrates, there are two main types of : muscle acins (including skeletal, cardiac and smooth muscle actins) and non-muscle (or In chicken, the actin multigene family encodes 10 - 12 isoforms, each cytoskeletal) actins. In organisms such as yeast, which contain only a single exhibiting distinct pattens of temporal and spatial regulation during the actin gene, the sequence is more similar to vertebat non-muscle genes, so ontogeny of the organism. The molecular mechanisms which control this is considered to be the ancestral state. The actins expressed in muscle actin gene expression during smooth muscle development are currently to cells of invertebrtes have aditionally been considered be non-muscle-like being addesed by our laboratory. A smooth muscle y-actin (SMGA) also. A number of scenarios have been to explain the evolution of proposed was obtained the muscle actin genes from a non-muscle ancestor. We have re-examined these isoform specific molecular probe previously through competing hypotheses within a phylogenetic framework. We compared the isolation and charcterization of full length cDNAs. We have used this amino acid sequences of more than 30 different actin genes, including probe for Southern blot analyses of EcoRI digested chicken genomic representatives from a number of different animal phyla. Our results suggest DNA which revealed a single hybridizing band of- lOkb in size. These that the gene duplication that led to the chordate muscle and non-muscle genes data suggest that SMGA is a single copy gene. The structure of the gene the before echinoderms and chordates occunred eariy in deuterostome lineage, was subsequently determined through the isolation and sequencing of diverged, and may even have occurred before the protostome-deuterostome Chain Reaction The split. Our results support one recent hypothesis that suggests that insect genomic clones and Polymerase (PCR) products. muscle actins arose independently from an arthopod cytoskeletal actin chicken SMGA gene contains 9 exon segments separated by 8 introns, ancestor, and are therefore convergent with chordate muscle actins. Contrary and the exon/intron boundaries of the chicken gene are identical in to another recent hypothesis, our resuts indicate that the gene duplication that placement to that of the human SMGA gene. Examination of the -2.3kb led to the different chordate muscle actin types (skeletal, cardiac and smooth 5' flanking sequences adjacent to exon reveals multiple CArG and E- muscle) had not occ rred at the time that Urochordates and Vertebrates box elements which may participate in the regulated expression of the muscle actin types are seen in Urochordates, diverged. Although different These domains are tested for these are probably the result of a gene duplication that occurred within the SMGA message. potential regulatory being Urochordate lineage, and are not reflected in other types of Chordates. their promoter activity in primary cultures of embryonic chicken gizzard cells. (Supported by Grant AL-G-91008 from the Am. Heart Assn.) Monday. Gene Structure II (1110-1115) 191a

1110 1111 MOLECULAR CLONING OF THE S' END NON-TANDEM REPEAT CLONING AND ANALYSIS OF A COMPLETZ GENZ ZNCODING A HYALURONAN REGION OF A RAT MUC2 MUCIN GENE HOMOLOGUE: EVIDENCE RECEPTOR RHAQMM (( Yang, B., J. Zntwintle, C. Wong, S. Zhang, FOR B.L. Yang, M. Mowat, A.H. Greenberg and E.A. Turley)) EXPRESSION IN BOTH INTESTINE AND AIRWAY. ((H. Omori, Manitoba Institute of Coll Biology, and Dept. of Pediatrics, A.F. Dohrman, M. Gallup, D.J. Steiger, T. Tsuda, H. Kai, J.R. Gum, Jr*., Y.S. University of Manitoba, 100 Olivia street, Winnipeg, Canada, Kim*, and C.B. Basbaum)) Department of Anatomy and Cardiovascular R3E 0V9 Research Institute, University of California at San Francisco, CA 94143. We screened a genomic 3T3 fibroblast DNA library with a *Gastrointestinal Research Laboratory, Veterans Administration Medical Center, hyaluronan receptor RHAMM cDNA that encodes the cytoplasmic San Francisco, CA 94121. form of this protein (cRHAMM). Five clones were isolated and sequencing showed them to be derived from the same gene. One of thea, RHAMMG9, contained the complete coding sequence and Mucin is the major glycoprotein synthesized and secreted by epithelial goblet was used for detailed analysis. Northern analysis using and mucous gland cells of the mammalian respiratory and gastrointestinal tracts. RHAMMG9 and RHAMK cDNA as probes revealed the same 4.4 kb and Three rat cDNAs have been isolated from a rat intestine cDNA library by 1.7 kb RNA populations. In the genomic clone, using the screening with an upstream non-tandem repeat cDNA fragment of the human cRHAMMG9 as a reference, the coding sequence occurred as 9 exons spanning 12 kb DNA and interrupted by 8 introns. The MUC2 gene, SMUC 313 (Gum J.R. et al, 1992). Combined nucleotide sequence 9 exons in the coding region were 99, 216, 368, 147, 153, 97, an open for the 3 cDNAs contained reading frame spanning 4.5 kb encoding 179, 152 and 17 bp, respectively. A sequence repeated 5 times 1515 amino acids, including a translation start site. This amino terminal was contained in the largest exon (368 bp). The two HA sequence contains a non-tandem repeat domain enriched in cysteine (1366 binding domains described previously by us (Yang et al, 1993, residues) followed by an irregular tandem repeat domain (149 residues). J. Siol. Chem. 268, 8617-8623) are separated by a large (2.2 Homology with the human protein is 85% in the non-tandem repeat domain kb) intron. 5' upstream regulatory elements have also been identified and are consistent with this gene being tightly and 37% in the irregular tandem repeat domain. Primer extension analysis regulated by cytokines. Research was supported by the suggested that the transcription start site occurs at 155 bp or 30 bp upstream of National Cancer Institute, of Canada with funds from The translation initiation. Northem analysis revealed a message size of about 12 kb Terry Fox Marathon of Nope (E.T. and A.G.) and the MRC of in intestine and airway. Exposure to LPS induced a marked increase in the Canada to E.T. amount of mRNA corresponding to this gene in rat airways. In situ hybridization demonstrated expression of the rat MUC2 gene homologue in tracheal submucosal glands as well as colonic goblet cells. The 5' flanking sequence of the rat MUC2 gene has been partially sequenced and exhibits a putative TATA box, CAAT box, SPI binding site, API binding site, and NFkB binding site. (Supported by HL24136+HL43762)

1112 1113 TARGETED DISRUPTION OF A VOLTAGE-GATED POTASSIUM CHANNEL GENE CLONING OF A NEW MEMBER OF THE PEROXISOME IN EMBRYONIC STEM CELLS. ((C.S. Ho and R.H. Joho)) Department of Cell Biology PROLIFERATOR ACTIVATED RECEPTOR (PPAR) FAMILY FROM and Neuroscience, The University of Texas Southwestern Medical Center, Dallas, MOUSE LIVER. ((Y.J. Zhu, K. Alvares, M.S. Rao, and J. K. Reddy)) TX 75235. Department of Pathology, Northwestern University Medical School, Chicago, Potassium (K+) channels are ubiquitous membrane proteins encoded by a diverse IL 60611. (Spon. by S.I. Roth.) multigene family. Each K+ channel gene exhbits a unique pattern of expression. In our previous studies in rats, we showed that two different votage-gated K+ channels Peroxisome proliferators are a diverse group of chemicals that elicit (Kv1.6 and Kv3.1) were preferentially expressed in brain but not In liver, lung, kidney, predictable pleiotropic responses in the liver of rodents. These agents cause heart and skeletal muscle. Expression of Kv3.1 was not detected at birth. Its mRNA rapid and coordinated transcriptional activation of specific genes, including was first detectable at postnatal day 11 (P1 1), and expression levels gradually enzymes for the increase during development. Similar findings were observed in mouse by RNase those encoding the responsible peroxisomal fatty acid oxidation protection assays. A recent report suggested Kv3.1 may code for the Mtype K+ by a receptor mediated mechanism. Recently, a mouse (mPPAR), rat (rPPAR), channel in mouse lymphocytes, and expression levels are significantly increased in human(NUC1), and three related receptors from Xenopus (xPPARa, xPPAR[, mice with autoimmune diseases. To study the biolgical signiicance of Kv1.6 and and xPPAR) were found to be activated by peroxisome proliferators. We Kv3.1, we are attempting to generate mice with null mutions at these loci by now report the cloning of a new member from mouse liver which we designate recombination in embryonic stem cells (ES cells). Twooverlapping phage homologous mPPAR'y. mPPAR^y is closely related to xPPARy with 75% identical amino clones containing a total of 17 kb genomic sequence encoding Kv3.1 were isolted showed only 55 from a129 mouse genomic DNA library. A DNA stretch of 6kb surrounding exon 2, acids, while it % identity with mPPAR. The ligand binding and which encodes the first through sbith tranamembrane segment (S1 to S6) including the DNA binding domains are best conserved between mPPAR'y, mPPAR, and putative pore region (H6), was used for gene targeting. A neomycin resistance gene xPPAR'y. mPPAR'y was able to impart peroxisome proliferator responsiveness (neo) was inserted after S2. For the double selection protocol, a herpes simplex virus to the promoter of peroxisomal bifinctional gene, which encodes the second thymidine kinase gene (tA) was pIeced at the 5' end of homology. Linearized enzyme of the peroxisomal fatty acid [-oxidation. Northern blot analysis constructs were transfected into ES cells by electroporatbon. neo or neoe/tK colonles showed that in mouse, the mPPARy is expressed: liver> kidney> heart> were picked and analyzed by PCR. Homologous recombination events were found in 10 out of 484 neo colonies. PCR positive clones were expanded and the lung> testis> brain. The mPPARy was not significantly induced in liver and recombination events were independently confirmed by Southem blots using genomic kidney by ciprofibrate, a peroxisome proliferator. DNA. Two targeted ES cell lines have been chosen for blastocyst injection to create mutant mice. (Supported by NS-28407 to R.H.J.).

1114 1115 IDENTIFICATION AND CHARACTERIZATION OF ALPHA 1- IN cmli OF BnY MUlNU =PLM OI8 ANTITRYPSIN GENES IN MURINE STRAIN 129. ((D. Dixon, J. VIRUS.8 ((K. Mita S. Ichimura-L, and S.Ilada")) 'Div if Kofron, J. Rood, L. Snyder, R. Ciccarelli, and R. Biology, Natl. Inst. of Radiol. Sci., Chiba 263, Japan, Dept. Lirette)) Department of Molecular Biology, Sterling of Entomology, Univ. of California, Davis, CA 95616. Winthrop Pharmaceuticals Research Division, Collegeville, PA 19426 (Spon. by R. Ciccarelli) The baculovirus Boikx ggj, nuclear polyhedrosis virus (BeNPV) was found to encode a ubiquitin-like protein (v-ubi). A cDNA Alpha-1-antitrypsin (AIAT) is a major serum protease clone of the v-ubi gene was isolated from fat body cells of the inhibitor in humans and mice, and is a member of the infected with BeNPV serine protease inhibitor gene family. This protein is silkworm, B.jpori, and sequenced. The deduced acid produced primarily in the liver, and functions as an amino sequence of the v-ubi gene showed 76X and inhibitor of neutrophil elastase in the lung. In this 99E identity to those of mamals and iAutoralba californica NPV study, we have identified at least three distinct (Guarino, 1990), respectively. Two regions of v-ubi, amino variants of the AlAT gene in murine strain 129, using acids 15-31 and 53-57, differed significantly from the PCR on genomic DNA. A high degree of both intron and corresponding sequences found in mamalian ubiquitin consensus exon sequence homology among the three variants has sequences. Northern blot analysis identified two transcripts been observed. Two of the three genes have conserved (400 and 600 nucleotide) in BnNPV infected fat body cells which Met-Ser reactive sites, while the third variant has a hybridized to a 30 nucleotide long probe with sequences Met to Tyr substitution at the active site. All three complementary to amino acids 20-29 of v-ubi. The transcripts of the variants are transcriptionally active, as arose from two transcriptional start sites and were transcribed evidenced by the isolation of gene-specific cDNA at similar levels. mRNAs transcribed from the v-ubi gene were fragments through RT- PCR of mouse liver RNA. The detected in fat body cells at 3 days post BmNPV infection, relative levels of transcription of each variant are indicating that the v-ubi gene was a late being determined, and the genomic organization of this IJIIQUITIN-L= baculovirus gene. The level of host ubiquitin gene aRNAs increased gene family is being explored. This following study BuNPV both in fat body demonstrates that there are at least three distinct, injection cells and posterior silk gland cells before the activation of the v-ubi gene. actively transcribed genes for AlAT in mouse strain Transcription of the tubulin gene, 129. beta however, was clearly depressed in fat body cells 3 days post BeV injection. The effects of expression of the v-ubi gene are now being characterized by mutagenesis experiments. 192a Gene Structure II (1116-1121). Monday

1116 1117 BM2, A NEW SINE OF BOMBYX MORI. ((S. Ichimural, STUDY OF AMERINDIAN EVOLUTION USING HUMAN SPECIFIC ALU INSERTIONS K. Mita1 and T.C. James2)) 'Division of Biology, National Insti- ((G.E. Novick, C.C. Novick, J. Garrison, A. Ramado, C. Menendez, M.A. Batzer, P.L. tute ofRadiological Sciences, Chiba 263, Japan. 2Department of Deininger, RJ. Herrera)). Department of Biological Sciences, Florida Intemational Molecular Biology and Biochemistry, Wesleyan University, University, Miami, Florida, USA. Middletown, CT 06459-0175. Alu is the largest family of Short Interspersed Repetitive Elements (SINEs) in humans. Each element is about 250 bp long and they are found distributed throughout primates in an excess of 500,000 copies per haploid genome. We previously described and characterized certain Human Specific (HS) The short interspersed repetitive elements (SINE) are consid- Alu insertions either as dimorphic, slightly dimorphic or monomorphic based upon: 1) PCR ered to be dynamic sequence components that may participate in amplification using primers directed to the unique sequences that flank the site of insertion of the different elements studied; 2) gel electrophoresis and scoring according genome gene rearrangements. evolution through mediating The to the presence or absence of an Alu insertion in one or both homologous chromosomes; Bomkx& mon genome contains a SINE which is a memberofthe 3) allelic frequencies cakulated according to Hardy-Weinberg equilibrium. Ourcurrent studies involvethemolecularcharacterizationof15Amerindian populations Bml family. In the present study, we isolated and characterized with respect to their allelic frequendes (ie. alleles containing or lacking the Alu insertion) ten independent clones of a new SINE from the B. mori genome. for four different HS Alu loci. The populations being studied include: Aleut (Alaska), Cree\Ojibwa (Canada),Sioux (USA), Moskoke (USA), Mayan (twopopulations, onefrom This SINE (named Bm2) is 323 bp long and has anA-rich 3' tail. Campeche, the other from Buctzotz, both from Mexico), Guahibo (Colombia), Arhuaco In some of the clones, short direct repeats flank the Bm2 ele- (Colombia), Kogui (Colombia), Guambiano (Colombia), Karitiana (Brazil), Surui (Brazil), Quechua (Peru), Inca (Ecuador) and Toba (Argentina). ments. The reiteration frequency ofBm2 in the genome is 104 as Our analysis is focus on grouping the populations understudy, identifying their origin, determined by dot blot analysis. Unlike the Bml SINE, the Bm2 and tracking their migrational patterns. Although different approaches are currently available for the analysis of biological element does not contain an internal split RNA polymerase Im evidence, the degree of variability observed using PCR amplification of Human Specific promoter and is not transcribed in vivo by RNA polymerase Ill. Alu inserions as DNA fingerprinting markers, has shown to cover a window of resolution that is left unexplored by marker either too static (eg. blood group typing) or In addition, the 3' region of this element shows 65% homology too dinamic (eg. VNTRs DNA typing). to the equivalent 3-terminal domain of primate Alu sequences. This research was supported by U.S. Public Health Service Grant RR08205 to RJ.H.

1118 1119 CLONING AND CHARACTERIZATION OF AN INSERTION MUTATION GENE TRANSFER AND EXPRESSION IN BOVINE ENDOTHELIAL IN THE DUSKY GENE OF DROSOPHILA MELANOGASTER. ((S.M. CELLS BY USING RETROVIRAL VECTORS. ((Y.Ma', F.02, L.P. DiBartolomeis)) Department of Biology, Millersville University, Millersville, Reynolds1, D.A. Redmerl, and E.S. Berry2)) 1Department of Animal and PA 17551. Range Sciences, and 2Veterlnary Science and Microbiology, North Dakota State University, Fargo, ND 58105. dusky (dy) is a gene located in the lOEl-2 region of the X chromosome of Drosophila. Mutations affecting this gene result in flies which have shorter wings than those of the wild-type. One dusky mutant, dy73, appears to have Retroviral-mediated gene transfer techniques currently have been its dusky gene interrupted by a stable insertion, probably a transposable developed to efficiently introduce genetic materials into eukaryoftc cells. element (M. Green, Mut. Res. 29: 77 (1975)). Northern and RNase Our objectlve was to establish a gene transfer and expression system protection assays indicate that the 4.2 kb dusky transcript is expressed in for bovine endothelial cells by using retroviral vectors. We first infected embryos and early through mid-late pupae but not in 2nd and 3rd instar larvae, a bovine endothelial cell line (BAE2B) and 3T3 cells with 1, 10 and 100 late pupae or adults (S. DiBartolomeis and R. Jackson, unpublished). pi supematant from producer cells (PA317) transfecled with pB2N Insertion of the putative transposable element in the dy gene appears to affect vector. The polymerase chain reaction products for Neo were detected the expression of the gene in dy73 mutants by increasing the length of the in BAE2B and 3T3 cells 48 hours after infection. BAE2B and 3T3 G418- transcript relative to the wild-type and by altering the developmental profile of resistant colonies formed at 6-12 days post infection. These results dy RNA expression: the dy73 transcript appears not to be subject to wild-type demonstrated that the NeoR gene can be integrated into the genomic it is in adult To developmental controls as present embryo through stages. DNA of bovine endothelial cells and 3T3 cells and expressed in both begin to how the mutation affects dy about 4.5 kb determine dy7 expression, cells consfftutively. We then introduced IacZ (E.coli) and luciferase of the approximately 5.5kb insertion has been cloned. A size selected plasmid genes to library was constructed and screened successfully for a clone containing a 4.5 (firefly) Into pB2N construct pNLacZ and pNLuc vectors, kb PstI junction fragmnent which contains both wild-type (dy) DNA and over 4 respectively. 5oth lacZ and luciferase genes were expressed in 3T3 kb of the foreign DNA. Analysis of this cloned element indicates that cells after transfection. Presently, we are modifying the vectors and restriction enzyme sites correlate well with those tentatively mapped to the evaluating expression of these genes in bovine endothelial cells. element by restriction fragment length analysis of genomic DNA. Attempts will be made to identify this foreign DNA as a transposon and to precisely characterize its insertion site in the dy gene by sequencing. Such information may lead to a better understanding of how dy gene expression is regulated and/or how the insertion of foreign DNA can deregulate gene expression.

1120 1121

INTRODUCTION OF FOREIGN DNA INTO EMBRYO CELLS BY USE OF THE FLAGO EPITOPE TO EXAMINE EXPRESSION AND PARTICLE BOMBARDMENT ((A.V.Zelenin, V.A.Kolesnikov, FUNCTION OF A SYNTHETIC GENE ENCODING A CELL I.A.Zelenina, M.L.Semenova, M.A.Rodova, L.K.Ernst, and A.V.Alimov)) Engelhardt Institute of Molecular SURFACE PROTEIN NI MAM4ALLAN CELLS. ((D. W. Bianca, B. L. R. G. Biology, Moscow 119846 and Moscow State University, Brizzard, Chubet and D. L. Vizard)) Department of Moscow 119899, Russia (Sponsored by Research, Development and Applications, Laboratory and Research Products Division, Eastman Chemical Company, New Haven, CT We showed formerly the possibility to use particle 06511 bombardment for transfecting different animal cells in culture [1], in organ explants and in rat in vivo A synthetic gene encoding a 68 amino (2]. Ballistic introduction of neo gene into develo- acid sequence has been ping fish embryo allowed us to obtain transgenic fish designed and prepared for expression in mammalian cells. [3]. In this work we introduced foreign genes into Features of this gene product indude a translational initiation mouse and human embryo cells. Mouse developing emb- codon, the signal peptide of the preprotrypsin gene for transport ryos at the 2-4 cell, morula and blastocyst stages and the8 amino acid N-terminal Flag epitope(N-Asp-Tyr-Lys-Asp- were used. Penetration of tungsten microparticles Asp-Asp-Asp-Lys-). This protein also features a 21 amino add into the embryo cell nuclei was found at all stages. glycosylated spacer Microparticles were found in 20 out of 22 analyzed region to lift the Flag peptide away from the blastocysts. In 12 cases the particles were located membrane, a 21 amino acid consensus sequence of aCass I integral in the inner cell mass. After beta-gal gene introduc- membrane protein and a short region of 3 arginine residues tion the enzyme activity was detected in 30-40% of designed to anchor the protein in the membrane, followed by two the inner cell After neo gene introduction mass. stop codons. The Flag epitope is used as a tool to demonstrate three transgenic mice were obtained. In experiments expression of this genetically engineered protein and to the with abortion material the plasaid DNA carry- study human structure-function ing neo gene was transferred through embryo skin and relationships of the amino acid sequence. geneticin-resistant fibroblast clones were obtained. (1] FEBS Lett 244, 65-67 (1989); [2] FEBS Lett 280, 94-96 (1991); (3] FEBS Lett 287, 118-120 (1991) Monday. Gene Structure 11 (1122) 193a

1122 ENGINEERED FIREFLY LUCIFERASE FOR IMPROVED IN VITRO AND IN VIVO REPORTING OF GENE REGULATION IN EUCARYOTIC CELLS. ((BA. Sherf and K.V. Wood)) Promega Corp., Madison, WI 53711. (Spon. by J. Grosch.)

Luciferase from the North American firefly (Photinuspysrls) utilizes ATP and 02 to catalyze the mono-oxygenation of beetle luciferin and the concomitant emission of a single photon. The cDNA encoding luciferase (luc) continues to gain favor as the gene of choice for reporting transcriptional activity in animal, plant and microbial cells. The luciferase reaction, modified by the addition of acetyl CoA to produce peraistent light emission, provides an extremely sensitive and rapid in vitro assay for quantifying luciferase expression in small samples of transfected cells or tissues. However, the growing interest in using luciferase as a real-time (ie., in wvo) reporter of gene exprssion in eucaryotic colls has raised concrns that i) the activity of luciferase, which is sequestered in peroxisomes, may suffer from Umited substrate availability, ii) high-level accumulation of luciferase in peroxisomes may adversely affect the physiological well-being of the host cell, and iii) palindromic sequences contained in luc may impede transcription of the reporter if recognized as binding domins by spurious regulatory elements in certain cell or tisue types. To addres these issues a number of changes have been made in the DNA and protein sequences of luciferase, resulting in a family ofeucasyotic reporter vectors that are superior to comparable vectors containing the native luc gene. The enhanced firefly luciferase, lucGL3, is engineered to be genetically neutral within the host environment, amenable to gene fusion designs, and efficient with respect to translation initiation and procession. The modified luciferase reporter enzyme is devoid of potential N- glycosylation targets to minimize post-translational modification and remains in the cytoplasm of host cells to optimize substrate availability. NIH3T3 cells transiently expressing the enhanced lucGL3 reporter gone demonstrat significantly higher luciferase specific activities than do cells expressing the native luc gene.

Tissue Specific Gene Expression II (1123-1126)

1123 1124 ZEBRAFISH RAR-y IS EXPRESSED IN THE REGENERATING CAUDAL FIN. EXPRESSION OF CYCL1N B IN DIFFERENTIATING LENS FIBER CEILLS. ((C.Y. ((JA. White, M.B. Boffa and P.M. Petkovich)) Cancer Research Gao and PS. Zelenka)) National Eye Institute, N.I.H., Bethesda, MD 20892 Laboratories, Departments of Biochemistry and Pathology, Queen's University, Kingston, Ontario, Canada K7L 3N6. Terminal differentiation of lena fiber cells is marked by chromosomal condensation and abrupt dissolution of the nuclear envelope. Since similar events during mitosis are catalysed by the cyclin B/p34(cdc2) linase, we have assayed for cyclin B mRNA and Retinoic acid (RA) is an important signalling molecule in vertebrate protein in embryonic chicken lens fiber cells. Significant levels of cyclin B mRNA Were pattern formation. The effects of RA are due largely to regulation of detected in lens fiber cells by reverse transcription and PCR amplification. The identity gene transcription, mediated by retinoic acid receptors and (RARs) of the PCR product was confirmed by sequencn In situ hybridization with sense and retinoid X receptors (RXRs). We have been using zebrafish (zf) as a antisense riboprobes showed that cyclin B mRNA is loalized in the superficial, nucleated model to study the role of retinoic acid in vertebrate regeneration and lens fiber cells only. On two dimensional immunoblots of lens fiber cell proteins, an sequence have isolated cDNAs which closely correspond in to the mouse antibody to chicken cyclin B detected a 48 kDa protein with a basic isoelectric point. The and human receptors. Zf RAR-y exhibits a high degree of structural cyclin B in lens fibers was apparently compkexed with p34(cdc2) kinase, since both protein and functional conservation. Zf RAR-y binds to a retinoic acid response were parially purified by affinity chromatography on p13-agarose beads. The partially element as a heterodimer with RXRs. In cotransfection experiments, zf purified proteins eluted from pl3-agarose showed kinae activity toward histone Hi, which RAR-y exhibits promoter selectivity similar to its mouse counterpart. was not affected by inhibitors of protein kInase A. Histone H kminase activity was Since zebrafish fins have the capacity to regenerate, we have used incrased approximately 2-fold by phosphatase dison, indicating that both actve and whole mount in situ hybridization to localize RAR-y transcripts in inactive complexes are present. These results demonstrate that cyclin B, normally only during and present normal and regenerating caudal fin tissue. The soft tissue between the expressed G2 S phase, is in terminallydifferentiating lens ce14s and support the possibility that the nuclear loss that accompanies differentiation may be rays express both in normal as well as regenerating fins. RAR-y catalyzed by the cyclin kinase. Intense expression of RAR-y is associated with the dense blastemal B/p34(cdc2) mesenchyme that forms at the ends of the bony rays at the level of amputation. This blastemal tissue is thought to undergo differentiation into the various cell types that will replace the lost fin.

1125 1126 ENHANCED DIFFERENTIATION OF PREADIPOCYTES FROM BROWN DEMETHYLATION INHIBITS TUMORIGENESIS AND INDUCES AN APOPTOTIC DEATH FAT TISSUE BY AN INSULIN SENSIZING AGENT, ((LA. Foollmi- PROGRAM IN ADRENOCORTICAL TUMOR CELLS (Yi) HARBORING A CONSTRUCT EXPRESSING SEQUENCES ENCODING THE Adams, and R.F.Kletzien*)), The Kalamazo, MI 49001. DNA METHYLTRANSFERASE GENE IN Upjohn Company, THE ANTISENSE ORIENTATION. ((R. A. Mackood, V. Bozovic, and M. Szyf)) Department of Pharmacology and Therapeutics, McGill University, Montreal, PG, Canada, H3G lY6. Brown adipose tissue (BAT) is the major site of facultative non-shiveing This project is supported by the NCI(Canda). MS. is a scientist of the NCI thermogenesis ma al. Th it drial uoupli protein (UCP) is unquely exprsedin BAT and functions as aproton channel to uncouple The involvement of DNA methylation in the oncogenic process is an open question. While many tumor call lines overexpreas the DNA melhystraferss gene, thereis no respiration from ATP synthesis. Pioglitazone is a novel antidiabetic direct evidence that this over expression is causative to the transformation process. To compound which lowers bloodglucos and in vwo by'increing triglycerides address this basic question we have attempted to directly mod lats the level of DNA imulin sensitivity intarget tissue. We have obrved that treatment of methylransferase gene expression in a murins adrenocort tumorcell lineYI. YI diabetic rodents with piogiitazon resulted in a substantial inmrease in BAT calls were transfected with a chinoric construct expresittdp1 from theS' of the DNA mass. To explore whether piogitasone oulddirectlyeffectBATgrowth and methyltransferase gene in the antisense orientation. CIlls that expressed the antisense construct demonstrated an alered pattern of DNA methylation at specific genes as diferentiation, a primary culture system of BAT preadipocytes was by Mspl/Hpall analysis as well as a general decreasein overall genomic developed. Our work hasestablished that the drug can directly enhance judged methylation as judged by nearest neighbour analysis and Hpall digestions. These celas difentiation of thes cells as by in logy monitored chane ellularm also showed distinct morphological alterations andbat the ability to growin an anchorage andbiemical changes in lipogenesis. Cultures mintainedin serum-fre independent manner as judged by growthin soft agar assays. The transfected clones medium wee used todistingui the pree contribution of various agents were shown to have a sinificant decrease in tumorigenicity in vivo compared to the to thedifentiton proess. Insulin appeared to be the prime effector of parental Ins as wet asoelis tranfdcted with control vector equences, when injected into syngeneic mice. Moreover, when the tumors that aross from cells diffetiation while ether piogtaxone or T, were found to enha LAFl expressing the antisense construct were analysed by northern analysis they were shown to haveiost insuli effects. To'examine posible ef tofthe drugangeneexprssicn, expression of the antisna oonstruct. In vitro, calls expressing the antisense construct we asessed the effct of drug treatment in vio and in vitro on the showed increased serum requirements, density restricted growth and induction of an exprsn of mRNA encoding UCP. We found a 2-fold increase in UCP apoptotic death program upon serum deprivation as judged by elctron mirosoopy and mRNA inBAT fom diabetic rodents treatedwithpioitaone. Piogitae observation of the characteristic130bp Internudcosomal ladder. These resuks provide us with first that DNA is In an in vitro produced a 22-fold increase in UCP mRNA. These studies demon- the evidence methylation actively involved oncogenic transformation. strate that pioglitazone directly enhan the growth and tiatio f BAT, which may be responsible for some of the metabolic effects of thi agent in animal models of diabetes. 194a Tissue Specific Gene Expression II (1127-1132). Monday

1127 1128 SWITCHING OF PARAMECIUM SURFACE ANTiGEN TYPES BY BINDING OF EFFECT OF TGF-BETA AND IL-1 ON THE EXPRESSIONS OF TWO CYCLOOXY- REGULATORY RNP TO INTRINSIC CELL MEMBRANE PROTEINS. ((R,Conning. GENASE GENES IN HUMAN AND BOVINE CELLS.((B.A. Jackson, D. John- W. Rumble, C. Uchtenberg and 1. Flmger)) Deportment of Biology. son*, C.E. Ristaino*, and P. Polgar)) Dept. of Biochemistry, Haverford College. Haverford, PA 19041. Boston Univ. School of Medicine; and Biotechnology Program, Massachusetts Bay Community College*, Wellesley Hills, Mass. Surface proteins in a number of unicellular eukoryotes exist in aternative forms that ore serologically distinguishoble. In Paramecium the repertoire Experiments were conducted to determine and compare the roles consists of obout o dozen antigens. only one of which is detectoble at a of Cyclooxygenase (COX)l and (COX)2 in TGF-Beta and IL-1 acti- porticulor time. Chonges In growth conditions elicHreversible swiching of vated prostaglandin (PG) synthesis in human lung fibroblasts these serotypes. We have been testing a model for this mutuol exclusion (IMR 90) and bovine pulmonary artery endothelial cells (BPAEC). which proposes that for every gene coding for an antigen there exists an Results show that TGF-Beta increases the steady state levels Inhibitor thot prevents tronscription. mRNA production is determined by the of (COX)l and (COX)2 messenger RNA in both IMR90 and BPAEC. localzation of individuol Inhibitors. If the site is nuclear, the gene controlled Time course experiements showed that in IMR90, TGF-Beta in- is not transcribed; iW bound to the antigen tself on the surfoce. a messoge creases within 4 hours, the level of a 5.5 kb COX message that.. can be tronscribed. We have partiolly purified putotive inhibitors, cohybridizes with the 2.7 kb COX1 message, which level increa- ribonucleoproteins of about 57 kD MW. which can switch serotypes and ses in 12 hours. IL-1 increases the level of COX1 message in bind to surfoce antigen and to 48 kD MW Intrinsic cell surfoce protein. RNA IMR90 but had no effect on the COX1 message in BPAEC. Neither apparently bridges an Inhibitor and the surface. Immunofluorescence TGF-Beta or IL-1 affected the COX2 message in either cell type. shows that RNase and proteinose K strip away the 57 kD RNP. leaving intoct Determinations of PG synthesis under conditions where the role paromecIa free to reoct with 48 kD antisera. ELiSAs with mixtures of isolated of phospholipase A2 is bypassed were conducted in conjuction 57kD and 48kD proteins (and their respective antisera) suggest that 48kD with these experiments. In IMR90, IL-1 increases the synthesis molecules have both a specific binding site (reacting with only one kind of of PGE2 under basal conditions and in response to bradykinin. Inhibitor) and also sites that bind with all Inhibitors. The relevonce of these TGF-Beta only increased PG synthesis in combination with IL-1. observations to switching and to regulation of gene transription will be Our results illustrate that COX1 acts as an inducible gene in discussed. both IMR90 and BPAEC.

1129 1130 MOLECULAR CLONNG AND CHARACTERIZATION OF BOVINE CONGWUTNIN. ANALYSIS OFN-ACETYLGLUCOSAMINYLTRANSFERASE V MRNA ((L.S. Liou, R. Sastry, K.L. Hartshom, Y.M. Lee, T.B. Okarma, A.l. Tauber IN MAMMALIAN TISSUES AND CELL LINES. ((G.-S. Perng, M. and K.N. Sastry)) Departments of Medicine and Pathology, Boston Shoreibah#, L Margitch+, M. Pierce# and N. Fregien+)) #Department of University School of Medicine, Boston, MA 02118 and Applied Immune Biochemistry, University of Georgia, Athens, GA 30602. and +Department Sciences, Inc., Santa Clara, CA 95054. of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, FL 33136. The bovine conglutinin (BC) is a member of a subgroup of the C-type vertebrate lectins called "collectins'. The other members of this V the addition of are the and surfactant A & D. N-Acetylglucosaminyltransferase (GlcNAc-T V) catalyzes subgroup mannan-binding protein proteins in a to the c(6-linked mannose of N- BC is found in the serum and appears to play a role in first line host N-acetylglucosamine 0(1,6) linkage linked chains on glycoe .Tis defense. Previously we have shown that purified BC can Inhibit glycan 0(1,6)N-acetylgluuaMine hemagglutination and neutralizes the infectivity of influenza A viruses. provides the prefemred site of addition of poly-N - 11_ sequence BC opsonized Influenza A viruses also show enhanced ability to stimulate We have recently cloned and sequenced a cDNA contaning the entire coding hydrogen peroxide production by human noutrophils. In order to sequence of GlcNAc-T V. In order to deermine which tissues express V mRNA we have done RNA blots and PCR facilitate studies on the structure-function relationships of this protein, GIcNAc-T gel analysis using RNA from sevcel rat tissues. These experiments show tht the GlcNAc-T V we have isolated and characterized a full length cDNA encoding BC. BC mRNA is 7.5 kb in and the levels of GlcNAc-T V cDNA was cloned from a bovine liver library and the nucleotide sequence about length highest in are seen in of 1519 base pairs was determined. The longest open reading frame mRNA are found brain and thymus. Lower levels intestine, encoded a 20 amino acid signal sequence and a protein of 351 aa. Analysis kldney, lung, heart and stomach. No GlcNAc-T V mRNA could be detected of the nucleotide and deduced amino acid sequence revealed a 87% and in muscle rliver RNAs. In addition, GIcNAc-T V cDNA was used to probe 78% Identity, respectively, with the sequences of another collectin, gel blots of RNAs from severl huanu and mouse cell lines. These results bovine surfactant protein-D (SP-D). A 94 bp stretch in the 5' showed that there are two RNAs, 7.5 and 9.0 kb, expressed in BW5147 untranslated region of BC cDNA showed 98% homology to the 3' end coding mouse lymphoma cells and in C2VC12 mouse myoblasts. Two RNAs, S and region of bovine albumin cDNA. Unlike SP-D mRNA, which is expressed 9kb, could also be detcted in HepG2 human hepatona cells, while only the in the lung, conglutinin mRNA expression was shown by an RNase 9 kb RNA could be seen in HL60 hunu lymphonia cels and only the 5 kb protection assay to be restricted to the liver. Isolation of the conglutinin RNA was detected in human A431 cells. These data suggests that the cDNA will allow molecular studies to determine the functions of various GlcNAc-T V gene is differentially expressed in nomal rat tissues and cell domains of this lectin. lines. Further, it appears that one mechanism of regulaton ofGlcNAc-T V may be at the level of RNA splicing.

1131 1132 HORMONAL REGULATION OF THE RAT CYSTEINE PROTEINASE INDUCTION OF CYPlAl, BUT NOT OF AN IMMUNOCHEMICALLY RELATED INHIBITOR (CYSTATIN S) GENE ((Orlando Chaparro, G. 52KD P450, IN ADRENALS OF 3-METHYLCHOLANTHRENE-TREATED GUINEA Warner Williams, and Phyllis A. Shaw)) Department of PIGS. ((V. H. Black, J. C. Witig, A. Wang, and P. Shaw)) Department of Cell Cell Biology and Anatomy, Mount Sinai School of Biology, New York University School of Medicine, New York, NY, 10016 Medicine, New York, New York 10029 A 52KD cytochrome P450, immunochemically related to CYP1A1,2,has been identified in guinea pig adrenals. To determine if this P450 Is affected by agents The cystatin S (cys S) gene contains several known to induce CYP1A1,2, guinea pigs were treated with 3-methylcholanthrene possible regulatory elements in its 5'-flanking (3MC). After two days, microsomes and RNA were prepared from the adrenais and sequence, including estrogen and glucocorticoid liver. Westem blots showed that two proteins (53KD and 56KD) Increased in liver responsive elements (ERE and GRE). The effect of microsomes of 3MC-treated guinea pigs. In adrenals low levels of the 53KD protein several steroid hormones on the expression of the were detected only after 3MC treatment. The 52KD protein, which reacted less Cys S gene was analyzed by PCR amplification of mRNA intensely with a number of polyclonal antibodies to CYPIA1,2, did not increase In from adult submandibular glands. Dexamethasone 3MC animais. RT-PCR, using oligonucotides specific for the 3' and 5' ends of induced high levels of Cys S mRNA in intact females, guinea pig CYP1Al, ampilfed a 1.55Kb cDNA from RNA of liver and adrenal tissue and lower levels in intact males. In from 3MC-treated animals. This cDNA was not detected In RNA from adrenals of adrenalectomized, castrated or ovariectomized untreated animals. Restriction enzyme digest paHtems of the 1.55Kb PCR animals, dexamethasone treatment resulted in no products were identical. Sequence analysis confirmed that these cDNAs CYPIAI. The to metabolize 7- change in Cys S mRNA levels. Beta-estradiol did not corresponded to guinea pig capacity ethoxyresorufin, an enzyrnatic activity usually associated with CYP1Al, inrewased 6 induce Cys S mRNA in intact animals, but induced Cys fold in liver microsomes of 3MC-treated guinea pigs. Metabolism of 7- S mRNA to a very high level in adrenalectomized, ethoxyresorufin occurred in adrenal micrsomes, alfthough at a much lower level, castrated or ovariectomized rats, which was reduced and was enhanced 3-4 fold by 3MC. Ethylmorphine demethylation, an activity when combined with dexamethasone treatment. correlating with the 52KD protein, occurred to a greater extent in adrenal than In Testosterone induced Cys S mRNA in intact males; liver microsornes and was not aitered by 3MC treatment. This study shows that this effect was amplified by adrenalectomy and CYPI Al can be induced in guinea pig adrenals by 3MC, but that it is quite distinct castration. In contrast, the induction by the from the Immunochemically related 52KD protein, In size, Intensity of beta-agonist isoproterenol (IPR), a known inducer of immunoreactvity, associated enzyme activity, and response to 3MC. (Supported the Cys S gene, does not appear to be affected by by NIH DK 39671 and HL 48476) the steroid hormones. These data indicate that glucocorticoids and sex hormones play a role in regulation of the rat cystatin S gene. Monday. Tissue Specific Gene Expression 1I(1133-1138) 195a

1133 1134 INVOLVEMENT OF THE GATA-3 TRANSCRIPTION FACTOR ISOLATION OF AP-1-SITE BINDING FACTORS FROM A PROGESTERONE- IN TROPHOBLAST-SPECIFIC EXPRESSION OF THE MOUSE INDUCED, SUBTRACTED PRIMATE ENDOMETRIAL cDNA LIBRARY. PLACENTAL LACTOGEN-I GENE. ((Y.K. Ng, J.D. Engel, and D.I.H. ((C.I.Ace and W.C.Okulicz)) Dept. Ob/Gyn, University of Linzer)) Department of Biochemistry, Molecular Biology, and Cell Biology, Massachusetts Medical School, Worcester, MA 01655. Northwestern University, Evanston, IL 60208 The female sex steroid progesterone (P) is essential for the The mouse placental lactogen-I (mPL-I) gene, a member of the prolactin/ normal development and maturation of the primate endometrium. growth honrone gene family, is expressed specifically in placental In order to identify P-dependent genes, we used a powerful trophoblast giant cells. Synthesis of mPL-I initiates at the peri-implantation subtractive hybridization method with cDNA prepared from stage, and peak mPL-I serum levels are achieved at mid-gestation. This endometrial tissue. Estrogen (E)-dominant cDNA (EcDNA) and P- hormone binds to the same receptor as prolactin, and functionally replaces dominant cDNA (PcDNA) were prepared from days 9-13 and days prolactin during mid-gestation. Our previous studies of the mPL-I gene 21-23 of artificial menstrual cycles respectively. The two defined a 274 bp promoter as sufficient for directing maximal expression classes of cDNA were ligated to EcoRI adaptors and amplified specifically in trophoblast cells. In addition to two AP-1 sites, sequences by PCR with an adaptor-complimentary primer. EcDNA (driver) between -242 and -188 were also found to be critical for trophoblast-specific was used to deplete PcDNA (target) of common sequences by promoter activity. Inspection of these sequences reveals a consensus binding multiple rounds of subtractive hybridization. Analysis of the site for the transcription factor GATA-3, and we find that GATA-3 does subtracted PcDNA by quantitive PCR revealed that it lacked indeed bind to this region of the mPL-I promoter. Transfection of an common sequences (e.g. actin and laminin) and contained P- expression construct for GATA-3, but not a mutated form of GATA-3 dependent sequences (namely placental protein 14). Subtracted lacking the zinc finger domain, results in increased transcription from the PcDNA fragments were cloned into a bacterial expression vector mPL-I promoter in trophoblast cells. Furthermore, introduction of the and individual clones sequenced. By comparing expression GATA-3 expression construct into non-trophoblast mouse L cells activates levels in original E- and PcDNA, approximately 60% of the the co-transfected mPL-I promoter, which is otherwise inactive in this cell subtracted clones are P-inducible or dependent. The background. The relevance of GATA-3 to mPL-I gene expression is also subtracted library was screened for transcription factors suggested by the detection of GATA-3 (and the closely related GATA-2) belonging to the AP-1 family. Fusion proteins induced from mRNA in the placenta, with the amount of the GATA-3 (and GATA-2) solubilized colonies were denatured, renatured and probed with mRNA varying during gestation in a pattern similar to that of mPL-I mRNA. a double-stranded, labelled AP-1 site under protein/DNA- Based on these observations, we speculate that GATA-3 (and GATA-2) may binding conditions. The procedure identified 6 AP-1 site activate additional genes in trophoblast cells, and that these factors may binding clones that are currently being characterized. therefore be critical regulatory components in trophoblast cell differentiation. (Supported by HD 20290).

1135 1136 MUTATIONAL ANALYSIS OF THE OSTEOCALCIN GENE PROMOTER TRANSCRIPTIONAL REGULATION OF A SQUAMOUS CELL INDICATES THE PRESENCE OF OSTEOBLAST SPECIFIC HELIX-LOOP- MARKER, THE SPR1 GENE, IN CONDUCTING AIRWAY HELIX (HLH) TRANSCRIPTION FACTOR (S). EPITHELIUM. ((Y. J. Chuu, S. Reddy, G. An, D. K. Ann, and R. Wu)) ((M. Tamura, J. Wozney# and M. Noda )) Dept. of Molecular Pharmacology, California Regional Primate Research Center, University of California, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 101, Davis, CA 95616 Japan. #Genetics Institute, Cambridge, MA, 02140. (Spon. by T. Yoshida) The small proline-rich protein gene (sprl) is a marker whose expression is Several members of the HLH proteins have now been reported and almost frequently associated with squamous cell differentiation. The expression of every member of this class has been implicated in transcriptional regulation in sprl gene in conducting airway epithelium is down-regulated by vitamin A cell type deterrnination and differentiation. To elucidate whether the members and up-regulated by phorbol 12-myristate 13-acetate (PMA) and cyclic of the HLH protein family are involved in controling an activity of osteocalcin AMP. We have recently isolated and characterized the genomic structure of (OC) promoter in osteoblasts, we examined the effects of overexpression of the human sprl gene (An et al., J. Biol. Chem. 268: 10977-10982, 1993). DNA trans-dominant negative regulator, Id-l. Transient cotransfection of the pOCA- sequence analysis revealed that the 5-flanking region of human sprl gene CAT, which contains 1.2 kb (-1094/+147) promoter region of rat OC gene contains an AP-1 binding site (TGAGTCA) at -141, an AP-1-like binding fused to a CAT reporter gene, with an Id-I expression vector resulted in site (TGAATCA) at -111, and a cyclic AMP-responsive element (CRE)-like suppression of CAT activities in ROS17/2.8 cells. Similar suppressive effect of (TGAGGTCA) at -588, relative to the transcnptional initiation site. To Id-l vector was observed when pOCCB-CAT (-199/+64) was used as a further understand the transcriptional regulation of this gene, chimeric reporter in ROS17/2.8 cells, but not in fibroblasts. This region contains two E constructs containing various deletion DNA fragments of5-flanking region box sequences (EOCI and EOC2). In ROS17/2.8 cells, basal transcriptional and the chloramphenicol acetyltransferase (CAT) reporter gene were used to activity was reduced when we used mutant CAT construct in which EOC1 was transfect both primary airway epithelial cells and the immortalized human destroyed. These observations imply the presence of transcription factor(s), airway epithelial cell line, BEAS-2B. It was observed that CAT activity in which could be blocked by Id-I protein, and would enhance the transcriptional transfected cells is elevated 5-7 fold by PMA and 3-5 fold by co-transfection activity of the OC promoter through interaction with EOCI sequence. Gel with c-jun expression plasmid DNA. However, CAT activity is more than retardation experiments indicated that factors in nuclear extracts from additively elevated (20-26 fold) by both PMA and c-jun expression ROS17/2.8 cells specifically bound to EOC1 sequence. This E-box binding treatments. RNase protection assay demonstrated that the increase in CAT activity was also found in the nuclear extract of C3Hl0T1/2 cells treated with activity was due to an elevation of CAT mRNA in transfected cells. rhBMP-2 but not in that of non-treated C3HIOTI/2 cells. Our findings Furthermore, the genomic footprinting analysis indicated that the AP-1 suggested that the activity of rat OC gene promotor would be regulated by binding site at -141, was protected in PMA-treated cells. These results certain osteoblastic E box binding proteins, involved in the osteoblast-specific suggest that sprl gene expression is activated through a PMA/c-jun expression of OC gene and differentiation of osteoblasts. -mediated mechanism.

1137 1138

MODULAR ANALYSIS OF A CONSERVED REGION WITHIN MAMMALIAN CLONING AND CHARACTERIZATION OF NOVEL TEF-1-RELATED HISTONE HlT PROMOTERS. ((S.A. Wolfe, D.A. Koppel, and S.R. FACTORS THAT BIND TO THE M-CAT MOTIF OF THE MYOSIN HEAVY Grimes)) Research Service, Overton Brooks VA Medical Center CHAIN 0 GENE. ((N. Shimizu, C.E. Yockey, G. Smith and S. Izumo)) and Dept. of Biochemistry and Molecular Biology, LSU Medical Molecular Medicine Unit, Beth Israel Hospital, Harvard Medical School, Center, Shreveport, IA 71101. Boston, MA 02215

The histone Hlt promoters from human, Rhesus monkey, Sprague- The A element in the rabbit myosin heavychain (HC) A promoter (-276/-263) contains the M-CAT motif, a element found in several muscle- Dawley rat, and New Zealand rabbit are highly conserved. A 39 cis-acting specific genes. The AeWment Isesentai for muscle-specific bp region between the Hl/AC box and the H1/CCAAT box (-92 and transcription ofthe myosin HCi gene. We have prevouslyidentified both muscle-specific -54 bp upstream from the transcriptional start site, and ubiquitous fators IA and A2, respectvely) that bind to the A element. respectively) has been examined with mobility shift and Sinoe the sequenceof the AeWmentiaiso vety similar to the GTIIC motf in methylation interference assays. The 18 bp element containing the SV40 enhancer, we screened a mouse cardiac cDNA library using an Hlt/CCTAGG motif (from -54 to -71) was compared to the human TEFI (GTIIC-binding factor) cDNA as a probe under low stringency corresponding 19 bp human consensus element in mobility shift conditions. we isolated three distinct clones: one encodes a mouse assays using nuclear extracts from the rat testis. The human homologue ofTEF (mTEF1) and the others encode mTEF1 -related proteins and2). The mTEF-i Is element differs at 3 residues and these differences are fTEFRI transcripts (12kb) ubiquitousl expressed. are indifferentiated responsible for an approximate fold increase in protein DNA tnree TEFR1 transcripts (7, 3.5, and 2 kb) expressed 5 skeletai myotubes but not in myobiasts orin non-muscie cells. screening binding affinity. Mothylation interference assays have By a Soi8 myotube cDNA library, weIsoated two distinct clones a and implicated 10 residues in rat (11 residues in the human (TEFR1 b) whichseems to be alternativelyspiced products. TEFRia encodes 427 consensus element) centered about the CCTAGG motif as being a.a. protein containing TEA motif (DNA bindingdomain) related to mTEF1. important for protein binding. The binding activity is found The amino acidsequenoe of TEFRI b Is the same as that of TEFRI aexcept exclusively in testis nuclear extracts and not in cytoplasmic for 43 a.a. Is deleted from next to the TEA DNA-binding domain. The Invtdro S100 extracts as determined by mobility shift assays. UV transcription/translatIon products of mTEF1, TEFRia and b specifically crosslinking experiments have indicated the Hlt/CCTAGG bound to the A elements. In gel mobility shift assay, the mobility of DNA- containing element binds to testis specific proteins having protein complxes of TEFRIb was simila to that of the musclespecific Al molecular masses of approximately 40 and 80 kD. Experimental factr. However the mobility of DNA-protein coplexes mTEF and TEFRIa was similar tothat of ubiquitous A2 factor. M-CAT motif is results using two other elements, one positioned at -79 to -92 Thus, to bound by a family of TEA domain factors, thatare in a and the Hl/GC consensus element at -70 to -81 will be containing expressed manner. presented. tissue-spocific 196a Tissue Specific Gene Expression II (1139-1142). Monday

1139 1140 COMPENSATORY GROWTH-MEDIATED MAMMARY EXPRESSIONOP SECRETORY COMPENTOP IMMUNOOLOBULIN A IN DEVELOPMENT AND GENE EXPRESSION. ((C.S. Park, and S.H. NASOPHARYNGEALCARCINOMA CELL. ((W. Chin, W.Y. Chm2, H.M. Husug, C.T. Kim)) Department of Animal and Range Sciences, North Dakota State Linl.2)) lahtiue of Bomdical Scincs Academi Si, Nankang Ilipei 11529, ad University, Fargo, ND 58105. 2Dqtpeg of Palhobgy, Natonal iw Univerity Hospital, Taipei, Taiwan, R.OC. (Spon. by L.S. Kao) The proper use of a time-dependent, nutritionally-regulated growth Secreory componen (SC)of Immnoglobuln A(IgA)isa ecepsm for IgA jointc (J regimen alters mammary differentiation, lactation and possibly chi chai) and ditibuled on the bsola plma mmbran of IssairAl mucom, livay prevention of mammary tumorigenesis. The objective of this study was gland mnmnry gln epdmeL hIb cla whede SC sRNA proin al milk on to determine the extent to which proteinsynthesized depends expres in the napaynge cucisomn (NPC) cela and nohuy mucos epuelia the milk protein mRNA accumulation in mammary epithelial cells. In this expiment we used Southr bklt walyss lo check SC DNA sequence in NPC cell, Sixty weanling female rats were assigned to either a control or stair- in situ nucleic acid hybridaion to ideaf SC uRNA and -m o1 ceisto localize step energy restriction (SSER) regimen, an alternating 3-2-3-4 week SC pte in both NPC culure clb original biops scimen. PFr compisos, other scheme beginning with 40% energy restriction, followed by refeeding. cancer cell lines wer also included. It wa found dt our NPC coll lies and die odtro The SSER model was designed to induce compensatory growth of cancer cell lie such an hepato, cevical cciom and pumonary cainwm cell li, mammary gland during different developmental stages. Mammary al swed the .me hybridization bod psn, similar O te noma hunan periphal blood tissues obtained from pregnant and lactating animals were used for cell DNA, when different endonucleases wer usd to diges the DNAL In stu nuleic acid acini culture expenments and chemical composition analysis. hybridizaion in NPC culure celb rewaled dot most culte cell contained SC mRNA with Mammary tissues from the SSER group showed an increase in DNA, some cell contned more SC mRNA recti produt otes Immn icaL calizaion showd most culue cells conting reaton product o thir cysoplaum on RNA and protein contents while exhibiting a decrease in lipid content die cell surface. Te bopw paaffin sco of four ce litn showed dat some tun cells as to those of the control tissues. acinar cells in compared Mammary contained SC protein whLe ote clls wee neptive. Some munor livary gland and ductal culture from the SSER group exhibited a 20% increase in milk protein epidhelis also showed anti-SC reacion producL However, the squanu metaplstic epielia secretion over that of the control. RNA blot analysis revealed that 0- of n msopharynx were neptive. Simiarly, thc hepata cell alo expres SC proein, while casein mRNA accumulation in acini from SSER tissue was increased as HeLa cell and lung adenocarcioma were negative. These ruiinp sggest that SC proein is much as 30-40%. Our data confirms that nutritionally-mediated not exprssd in squmous metaplstic epithelis of nmopharynx and dt only some malignant compensatory growth and hyperplasia of mammary gland during early ransfonned umor cell expres SC prein, indiating dat only ursorned NPC tumo cella developmental stages (puberty to pregnancy) alter subsequent containing SC protein and can be infected by EBV if the EBV and IgA ni-EBV-VCA wer mammary gene expression and lactation. pfesent srrounding the tumorc els at the nme time. Similasly, the hepatona cell might also be infected by EBV if the EBV and IgA were available.

1141 1142 THE HUMAN a(2 INTEGRIN GENE PROMOTER: IDENTIFICATION TRANSFORMATION-RELATED GENE EXPRESSION IN SV40-iNFECTED HUMAN OF POSITIVE AND NEGATIVE REGULATORY ELEMENTS EPITHELIAL CELLS IS LINKED TO INTEGRATION OF SV40 ENHANCER/PROMOTER IMPORTANT FOR CELL-TYPE AND DEVELOPMENTALLY- SEQUENCES. ((G.T.Chen, R.Vasquez, and M.L.Stelnberg)) Department of Chemistry - RESTRICTED GENE EXPRESSION. ((MM Zutter, SA Santoro, AS Biochemistry Division, The City College of New York, New York, NY 10031 of and Medicine, Painter, XL Tsung, A Gafford)), Departments Pathology Infection of cultured human kerstinocytes by the oncogenic virus, SV40, results In School of St. MO 63110. Washington University Medicine, Louis, the establishment of permanent transformed cell lines. In these cells the timing of appearance of various transformed propertles divides the transformatIon process The a2p, integrin serves as a collagen receptor or a collagen/laminin Into early and late phases. In the lat phase changes In the exprssIon of several receptor, depending on cell type. Expression of the a2pl integrin is highly transformatlon-rlatd genes has been previously reported Including altrd pattems regulated during normal cellular differentiation and is altered during of fibronectin synthesis and localizatlon, ectopic expression of sImple epithelal carcinogenesis. We have previously demonstrated that increased expression genes and several novel cDNA markers of transformation (PNAS 82:8498- of the differentiation is a 8502, ECR 182:461). The altered patterns of gene expression require more than 150 a2Pi integrin during megakaryocytic consequence cell generations to be fully manifest During this time period free viral DNA Is of increased a2 mRNA due to transcriptional activation of the a2 integrin gradually eliminated and only viral squences integrad Into host cell DNA at gene and that the decreased expression of the integrin in breast approximately 1-5 genome equlvalentsicell are retained. Since cells are never cured adenocarcinoma is due to decreased steady-state levels of a2 mRNA. We of the viral sequences, the viral early region Is apparently still required to maintain now report the identification and characterization of the 5' flanking region of indeflnite growth. We have cloned and sequenced the Integrat viral sequences from the a2 integrin gene. The 5' untranslated region of the a2 mRNA extends genomic libraries derived from a clonal subline of long term SV40 transformants. A 129 bp 5' to the site of translation initiation. The promoter region lacks small Integrated segment of the virus that consists of SV40 bases 15 - 478 which TATA and CAAT boxes but contains an abbreviated initiator sequence and contains the SV40 early promoter, 72 and 21 base pair repeat enhancer elements and sites. Consensus sites for AP-1 and AP-2 the thrid T antigen binding site, but none of the SV40 coding regions was found at a six Spl binding binding location about 1.8 kb distal to a full length Integrant and In an orientation which a Pu.1 and two motifs with complexes, a GATA box, box, palindromic would place coding sgments in the flanking human sequences under the control of potential to bind the estrogen receptor are also presenL A 961 bp fragment the SV40 early promoterienhancer. Sequence data from about 2 kb of the leftward of the 5' flanking region directs expression of a reporter gene in T47-D flanking human sequences Indicates the exlstnce of several possible coding regions epithelial cells and in pluripotent hematopoietic K562 cells upon whose expression may be regulated by the Integrated SV40 enhancer elements. megakaryocytic differentiation. A series of deletion mutants identifies the Based on data from other, Independently derived cell lines, we propose that cells presence of both enhancer and silencer elements necessary for cell-type and containing viral sequences integrabtd In this fashion represnt a highly transformed differentiation-specific gene expression. subpopulatlon which come to predominate In the population by a process of selection over long periods of time In culture. The dat also suggest that Integration of the promotertenhancer element per se may be required for the altered gene expression seen In long term viral transformants. Ribonucleoproteins and RNA Processing (1143-1144

1143 1144 FUNCTIONAL DIFFERENCES BETWEEN OOCYTE AND A DNA ELEMENT FOR rRNA GENE TRANSCRIPTION TERMINATION IN SOMATIC 5S RNA IN XENOPUS OOCYIES. ((LA. Allison and ARTEMBA. ((T. Luo and J.C. Vaughn)) Department of Zoology, Miami M.T. North)) Department of Zoology, University of Canterbury, University, Oxford, OH 45056. Christchurch, New Zealand. The comparative sequence approach can provide Insight Into iden- tification of DNA regulatory elements. We are using this method to Xenopus has two families of 5S ribosomal (rRNA) genes, oocyte-type identify such elements in ribosomal RNA (rRNA) transcription ter- mination. of recombinant and which are under control. In Screening complete libraries constructed somatic-type, developmental Xenopus for the related brine shrimp species Aremia frandsc2na (A.f.) and oocytes, both oocyte-type and somatic-type 5S rRNA are synthesized. A. rengSet (A.12.) has produced clones with complete rRNA Although somatic-type 5S rRNA has been shown to be incorporated gene repeat units. Vent polymerase PCR has also produced a 5.4 kb into ribonucleoprotein (RNP) storage particles immediately after fragment containing the entire intergenic spacer from A. p. Total synthesis, it has not been detected in the pool of ribosomes stored in cell RNA was isolated from each species, using developmental stages enriched for These RNAs were to or the oocyte. It was of interest to determine whether the six somatic- pre-rRNAs. hybridized cloned PCR-ampUfled end-labeled DNA fragments, for SI nuclease locali- specific substitutions alter the subcellular distribution of the 5S rRNA zation of the transcription termination site (TTS). This site is about molecule after microinjection into the oocyte cytoplasm. A greater 150 (in Af.) to 200 nucleotides (in A.-.) downstream of the 26S rRNA amount of microinjected NP-labelled somatic-type 5S rRNA was found coding sequence. Combining the SI method with DNA sequencing in the nucleus compared with oocyte-type 5S rRNA, after overnight has precisely localized the TIS 159 nucleotides downstream from the incubation. Somatic-specific substitutions increase the affinity for 26S sequence in A. f. Twenty-two nucleotides further downstream from TIS we have found a short conserved DNA tract: 5'- G-A-C-T-G- binding the protein TFIIIA in Wtro. However, within the oocyte, G-G -3' in A-f. The corresponding tract has been shown by others to microinjected somatic-type 5S rRNA preferentially bound to ribosomal be part of a larger element that is both necessary and sufficient to protein L5, forming 5S RNPs, as assayed by nondenaturing signal pre-rRNA transcription termination and 3'-processing in polyacrylamide gel electrophoresis. In contrast, microinjected oocyte- Xen=W and mouse. We are now working to obtain sequence for the type 5S rRNA preferentially bound toTFUIA, forming 7S RNP storage homologous region in A. R., from which it should be possible to use assays showed that the comparative approach to identify the complete DNA regulatory particles. Immunoprecipitation microinjected element in an arthropod. A general model for the role(s) of primary somatic-type 5S rRNA was assembled into 60S ribosomal subunits factors in rRNA gene transcription termination may then emerge. within the oocyte. Monday. Ribonucleoproteins and RNA Processing (1145-1150) 197a

1145 1146 TRANSCRIPTIONALLY ACTIVE 5S rRNA GENES ARE ASSOCIATED WITH THE IMMUNOFLUORESCENCE STUDES OF NUCLEOLIN DISTRIBUTION NUCLEOLUS. ((A.H.Bakken, J.Leu, and J. Meyer)) Department of WITHN AMPHIBIAN GERMINAL VESICLES. ((M.L. Rankin and P.J. Zoology NJ-15, University of Washington, Seattle, WA 91l95. DiMario)) Department of Biochemistry, Louisiana State University, Baton Rouge, LA 70803-1806. The nucleolus is the site of ribosomal RNA synthesis and the site of ribosome assembly. Four different rRNAs and some 80 Xenopus nucleolin was purified from a S100 cell extract of cultured kidney ribosomal proteins come together in this intranuclear organelle cells by DEAE chromatography and then poly[G] "affinity" chromatography. to be assembled into the small and large ribosomal subunits be- Nucleolin prepared by this two step procedure was considered pure by silver fore they are exported to the cytoplasm as the cellular machinery stain analysis, and those fractions containing only nucleolin were used for for translation of mRNAs. We report here an interphase associa- antibody production. The resulting rabbit serum, R2D2, labeled only the two tion of genes located on different chromosomes which results in Xenopus nucleolin proteins on Western blots versus a large complex mixture bringing their RNA-gene products together within the nucleolus ofcelular proteins. In double-labeling immunofluorescence experiments, both where they are assembled into ribosomes. A survey of the liter- mouse anti-fibrillarin (Mab72B9) and R2D2 labeled the dense fibrillar regions ature indicated additional types of RNA and proteins have been of the muldple nucleoli characteristic of the oocyte germinal vesicles (GVs). found in the nucleolus, e.g. U3 small nuclear RNA, nucleoplasmin, The granular regions of the nucleoli were lightly stained with R2D2 as fibrillarin, N038 etc. We have shown using three different types previously described for other anti-nucleolin antibodies. When the nuclear of analysis that some of the 5S rRNA genes are closely associated contents were allowed to completely spread, our anti-nucleolin serum labeled with and transcribing 5S RNA in the nucleolus in somatic cells granules that are much smaller than typical nucleoli. The bulk of the nuclear of the mouse (and probably in Drosophila and Xenopus as well). RNP material did not stain under these particular spreading conditions. When The 5S rRNA genes are located on different chromosomes from the spreading of Xenopus GV contents was restricted by increased magnesium other rRNA genes. Therefore, one must conclude that their assoc- concentrations, R2D2 weakly labeled much of the RNP material. In newt iation is a transitory one, that occurs only when both groups of GVs, R2D2 again intensely stained the dense fibrillar regions of the multiple genes are active. While the mechanism for this association is nucleoli, and lightly stained their granular regions. The main axes and lateral as yet unknown, the potential for co-regulation of their trans- loops of the newt lampbrush chromosomes were also labeled to the same cription is obvious and suggests an exciting new level of gene intensity as the granular regions of the nucleoli. myc-tagged nucleolin regulation, i.e. the intranuclear architecture of the cell. synthesized from injected mRNA showed similar extra-nucleolardistributions (This research was supported by NSF grants to A.H.B.). in Xenopus GVs after staining with anti-myc Mab 9El0. Our observations confirm that nucleolin is highly concentrated within the dense fibrillar regions of nucleoli, and they suggest that nucleolin distributes throughout the nucleus in lower concentrations. This work supported by NSF grant MCB-9204796.

1147 1148 N VITRO ASSEMBLY OF COILED BODIES IN XENOPUS EGG COILED BODY INDUCTION IN ESTROGEN-STIMULATED ROOSTER HEPATOCYTES. ((K. Brasch, D. Gallo, R.L. Ochs, T.W. EXTRACT. ((D.W.Bauer and J.G.Gall)) Department of Embryology, Stein, J. Teigen, and J. Williams)), Dept. of Biology, Carnegie Institution, Baltimore, MD 21210. California State University, San Bernardino, CA 92407 and Autoimmune Disease Center, The Scripps Research Institute, La Jolla, CA 92307 When incubated in an appropriate Xenopus egg extract, demembranated sperm nuclei become surrounded by a typical Coiled bodies (CBs), which appear to arise from the nucleolus and then to nuclear envelope to nuclear migrate the nucleoplasm, have and begin import proteins. Minute generated much interest recently, because they contain granules appear in the nuclei and gradually enlarge, reaching both nucleolus-associated elements and also components diameters of 2-3 pm. Bell et al. Cell Biol. 118:1297,1992) showed involved in pre-mRNA splicing. CBs are most prominent in a. hyperstimulated cells, including many cancers and steroid that these "prenucleolar bodies" contain several nucleolar antigens, target tissues. Using antibodies to the nucleolar including fibrillarin, but lack rRNA. We confirmed the presence of protein fibrillarin, to the CB-specific protein p80- fibrillarin and also found strong staining with antibodies against the coilin, and to human estrogen receptors, we have examined CB induction in rooster liver after primary estrogenic Sm antigen, trimethylguanosine, and coilin. By in situ hybridization stimulation. Animals are sacrificed 48 hr and 4 weeks with 3H-labeled probes we demonstrated the presence of U2, U3, U4, after a single injection of estradiol (20 mg/kg body weight), and after 1 week of continuous treatment. U7, and U8 snRNAs (Ul was abundant throughout the nucleus, Electron microscopy revealed two nuclear body making an assessment in the bodies difficult). These bodies thus morphotypes, one a classical CB and the other a hollow, contain constituents from three RNA processing pathways: pre- ring-like body. Double label immunofluorescence also identified two nuclear body populations, one labeled mRNA splicing, histone pre-mRNA 3' end formation, and pre-rRNA primarily for coilin and the other for fibrillarin, with processing. The components so far demonstrated broadly overlap approximately 251 overlap between them. Controls and 4 week post-hormone nuclei averaged 1.5 CBs/nucleus, those found in coiled bodies from mammalian tissue culture cells, whereas fully stimulated nuclei averaged 3.3 CBs/nucleus. suggesting that these structures are more closely related to coiled Western blotting results confirmed an increase in p80- bodies than to nucleoli, and may be identical to somatic coiled coilin in the 48 hr and continuous stimulation samples compared to the controls. Studies are currently underway bodies. By selective removal of snRNAs or proteins from the egg to assess whether either type of nuclear body contains extract, it will be possible to study the requirements for coiled body estrogen receptors. assembly in this system.

1149 '1150 COILED BODIES IN THE NUCLEOLUS OF BREAST CANCER CELLS. SPLICING OF RNA IN A MYOGENIC CELL ((R.L. Ochs, T.W. Stein Jr., and E.M. Tan)) Department of Molecular SYSTEM. ((Y.-C. Wang, A.R. Krainer, and D.M. Helfman)) Cold Spring and Experimental Medicine, The Scripps Research Institute, 10666 N. Harbor Laboratory, Cold Spring Harbor, NY 11724. Torrey Pines Rd., La Jolla, CA 92037. In rat and mouse, the mRNAs for two tropomyosin (TM) isoforms, 1-TM in Coiled bodies (CBs) are a special type of small round nuclear body, skeletal muscle and TM-1 in fibroblasts and smooth muscle, are composed of coiled fibers and granules, especially prominent in the generated through alternative RNA splicing from the same pre-mRNA of the 13-TM gene. In order to understand the regulation of nucleoplasm of highly active cells (Brasch and Ochs, Exp. Cell Res. 202: B-TM pre- mRNA splicing in skeletal muscle, we have analyzed in myogenic cells 211-223, 1992). Although no specific function has been assigned to the levels of certain splicing factors which are known to be involved in CBs, they contain spliceosome snRNAs and proteins, as well as the alternative RNA splicing of other genes. We have also been developing a nucleolar U3 RNA-associated protein fibrillarin. In the present study, we cell-free splicing system from a myogenic cell line, which will be used for have used antibodies to the coiled body-specific protein p80-coilin, studying the cellular factors regulatingB-TM pre-mRNA splicing. together with double-label immunofluorescence, confocal microscopy Western blot analysis was performed on whole cell lysates prepared from and immunoelectron microscopy, to examine the distribution of CBs in a the mouse BC3H1 muscle cells at different stages of myogenic number of different breast cancer cell lines. By immunofluorescence, all differentiation, using antibodies against splicing factors SF2 and U2AF, cell lines had prominent CBs in the nucleoplasm and several cell lines as well as polypyrimidine tract binding protein. Although all of these were present at appeared to have CBs within the confines of the nucleolus itself. Double proteins constant levels throughout the differentiation process, U2AF appeared to be more sensitive to a label immunofluorescence and confocal laser scanning microscopy proteolytic activity in muscle cells. For establishing the in vitro system, nuclear extracts were confirmed the nucleolar localization of CBs. Besides containing p80- prepared from BC3H1 cells according to the method used on HeLa cells, coilin, nucleoplasmic and nucleolar CBs contained fibrillarin and Sm with several modifications to optimize the system. The nuclear extracts proteins. By conventional and immunoelectron microscopy, nucleolar were then tested in an in vitro splicing reaction, using 32p labeled TM CBs appeared as discrete round bodies within the nucleolus in various pre-mRNA substrates as described previously (Helfman et al., Genes stages of 'maturation", but distinct from the normal nucleolar Devel. 2:1627-1638,1988). The results showed that the BC3H1 splicing components of granular component, dense fibrillar component, and system required more proteinase inhibitors and different salt conditions fibrillar centers. While the significance of finding CBs in the nucleolus of in the buffers for preparing the nuclear extracts when compared to the certain breast cancer cell lines is presently unknown, this represents the conditions used for the HeLasplicing system. The splicing patterns first report of CBs and Sm staining in the nucleolus. observed using the nuclear extracts made from undifferentiated and differentiated BC3H1 cells will be presented. 198a Ribonucleoproteins and RNA Processing (1151-1156). Monday

1151 1152 DIFFERENTIAL EXPRESSION OF JC VIRUS LARGE AND SMALL ALTERED POST-TRANSCRITIMONAL PROCESSING OF THE MRPLP T ANTIGEN mRNAs IN BRAIN TISSUES FROM PROGRESSIVE TRANSCRIPT IS RESPONSIBLE FOR EXPRESSION IN 3T3 CEULS. ((U. M. MULTIFOCAL LEUKOENCEPHALOPATHY (PML) PATIENTS WITH Malyankar. S. R. Ritling, and D. T. Denhardt)) Dqeunent of Biogical Scieces, AND WITHOUT AIDS. ((M. Ishaq and G. L. Stoner)) Laboratory of Experimental Neuropathology, National Rutgers,The Stae Univerity of New Jrey, Picaaway, 08854. Institutes of Health, Bethesda MD 20892. (Spon. by H. deF. Webster) MRP(misge regulated protee)P F(pma) is a glycop eis tht is se d into cel culue modium by 3T3 oel; sem stimulat for 8&12 boer enhances ct JC virus (JCV) causes PML, the fatal demyelinating expresson of the gene. On the ote hand, morteal mouse embryo fbroblub (MEPs), infection of oligodendrocytes, in up to 5% of AIDS patients. We have studied the expression of JCV from which dt 3T3 lnes deriv, ow anotundtecabk lev of expreion of early mRNAs (large T and small t antigens) in brain te gene shown by Nore bomts. On sceening the blo for aprcessed me tissues from PML patients with and without AIDS. usg two diffeentitro prbes, the unprcsd m_em is vible in the moral cell RNA-PCR using primers spanning both large and small Nuclrrun-on and tan truecuon expeimenta have also dshn tht the gene T-antigen introns allowed specific amplification of tcriptony Wehave uoed RT-PCR to resuts ad alternately spliced messages in the presence of acdve. confirm dte to quante contaminating DNA. Large T-antigen mRNA was detected the level of ascript in the MEF and 313 cells. We find dat the level of the in all PML and PML/AIDS brain tissues. However, unprocessdtanscript in MEFs is about balf tat in 3T3 cells Riboslme potecti brain tissues from 11 of 12 (92%) PML/AIDS patients assays of cells sftd with a constuct consisin of neary ft entie driv by also expressed small t-antigen mRNA, whereas only 8 CMV promoter indicat that te can be spliced in ME as well cells. of 13 (62%) with PML alone showed detectable levels Xt pe 313 of small t-antigen mRNA. These results suggest the Poly A RNA from 3T3 and MEF ces wa analyzed by RT-PCR and althugh the level following. First, the alternative splicing of JCV of proessd mesag was constently high 313 cells, the MEPs showed lele or no early message regulated by unknown mechanisms. is pmosesd mesc. We ar in the process of determining which egio of he tncript Second, the expression of small t-antigen may not be b meae essential for the pathogenesis of PML. Third, the might responsible for the destabilization of the in the MEF cells, or presence of HIV-1 may in some way influence the conversly for stabilizaon in the 33 celhl (Suppored by NIH AG 07972) splicing of T-antigen mRNAs and thereby promote JCV replication in the brain.

1153 1154 ABERRANT SYNTHESIS AND PROCESSING OF MYOTONIC SEQUENCE AND EXPRESSION OF TWO POLY(A) BINDING DYSTROPHY PROTEIN KINASE mRNA AS A RESULT OF A PROTEIN mRNAS IN MOUSE TESTIS. ((K.C. Kleene, M-Y. Wang, C. TRIPLET REPEAT EXPANSION. ((P. Carango, J.E. Noble, H.G. Hall, M. Cutler, and D. Shih)) Dept. of Biology, University of Marks, and V.L. Funanage)) Medical Cell Biology and Neurology Massachusetts, Boston MA 02125. (Spon. by B. Harrison.) Depts., Alfred I. duPont Institute, Wilmington, DE 19899 The important role of post-transcriptional gene regulation in mammalian Myotonic dystrophy is an autosomally dominanant inherited male germ cells prompted us to investigate the expression of the poly(A) disease in which system-wide abnormalities are caused by a tnplet binding protein (PABP), a protein which binds to the poly(A) tail of repeat expansion within the untranslated region of the myotonic 3' mRNAs and is believed to influence mRNA stability and translational dystrophy protein kinase (DMPK) gene. To determine the effect an cDNAs encoding two PABP mRNAs were expanded repeat region has on DMPK expressison, we have activity. isolated from mouse libraries. The deduced amino separated the chromosome 19 homologues from a 36 yr. old testis acid sequence of one (PABP1) is nearly woman with myotonic dystrophy into different cell lines by way of identical (98.7%) to human liver PABP, while the second (PABPt) shows somatic cell hybridization. Hybrid DM9101 contains the normal lower amino acid identity with mouse PABPI and human PABP, ca. 80%. DMPK allele (13 repeats), whereas hybrid DM1 115 harbors the Northern blots reveal that there is one major PABP mRNA species in liver, mutant allele (133 repeats). RT/PCR amplification of coding spleen, muscle, kidney and brain, while testis contains multiple size-variants sequences from the DMPK gene has shown both reduced levels of and much higher levels of PABP mRNAs than somatic tissues. The high primary DMPK transcripts and impaired processing of these levels and multiple PABP mRNAs are present in highly enriched meiotic transcripts in hybrid cell line DM1115. These findings suggest that and early haploid spermatogenic cells, but the PABP mRNA levels are the presence of a large number of repeats in the 3' untranslated much lower in late haploid cells. The vast majority of testicular PABP region of the DMPK gene reduces both synthesis and processing of mRNAs sediment slower than single ribosomes indicating strong DMPK mRNA, resulting in undetectable levels of processed DMPK translational repression. Reverse transcriptase-polymerase chain reaction mRNA from the mutant allele. assays show that the levels of PABPt and PABPl mRNA, respectively, are about lOOOX and lOX higher in testes than in somatic cells. These findings demonstrate the presence of a variant PABP mRNA in spermatogenic cells.

1155 1156 ANALYSIS OF THE ORGANIZATION OF METAZOAN pm.nRNA IN SITU HYBRIDIZATION COMBINED WITH METABOLIC PROCESSING AND INTRANUCLEAR TRANSPORT IN A NOVEL LABELING REVEALS RELATIONSHIP OF POLY (A) WITH NEWLY EXPERIMENTAL SYSM. ((Zuzana Zachar, Joseph Kramer, and Paul M. SYNTHESIZED TRANSCRIPTS. ((F. and R. H. Bingham)) Dept Biochemistry & Cell Biology, University of New York, Stony Brook, S. Fay*, K. L. Taneja NY 11794-5215 Singer)) Departments of Physiology* and Cell Biology, Univ. Massachusetts Medical School, Worcester, MA 01655 We have developed a novel system for analysis of organization of nuclear pre-mRNA metabolism based on the powerfil genetic system and giant, polytene nuclel of Transcripts were labelled in human fibroblasts over a 10-30 Drosophila. Among other things ourstdies show the following: minute time period by intracellular microinjection of 5- 1. Several lines of evidence indicate that pre-mRNAs move away from directly bromouridine triphosphate and detected by a fluorescein- template DNA after polyadenylation in an isotropic fashion and at rates consistent with diffusion. conjugated antibody (Wansink et al. J. Cell Biol. 122: 283-293, 2. This route of intranuclear movement is used by most or all pre-mRN. 1993). The same cells were then detected by in situ 3. The diffusional movement of pre-mRNAs is constrained by exdusion from hybridization to poly (A) RNA using an oligo dT probe directly condensed nuclear structures such as chromosomal axes and nucleoli. Thus, diffusion conjugated to Texas red. The conjugated probe provided better is restricted to a system of interconnecing danels referred to as the penetration of the nucleoplasm than indirect detection and extrachromosomal channel network or ECN. revealed a dispersed but patchy pattem of poly (A) labelling. 4. Components of the constituve splicing machinery are coconcentrated with pre- mRNA in the diffusional domain represented by the ECN. Images for each fluorochrome were obtained as a 3D data set 5. Details of our results indicate that retention of incompletely spliced pre-mRNAs and precisely aligned using fiduciary markers. Co-alignment is a function of the nuclear surface not of intranuclear structurs. revealed areas of overlap and non-overlap between recently In summary, we will argue that a simple model including (a)organization of nascent labelled (predominantly nascent) transcripts and poly (A), pre-mRNAs around template DNA, (b) channeled, intranuclear diffusion of representing predorminantly post-transcriptional RNA. polyadenylatedpro-mRNAs and splicing components, (c) simple properties of punctate Quantitation of the percent overlap as a function of nuclear substructures locatd within this diffusion domain and (d) discriminatin labelling or between mare ad incompletey spliced premRNAs by the nuclear surface can times actinomycin treatment reveals the kinetic relationship account for all available evidence concerning the behavior and organizaon of between new transcripts and nuclear poly (A). Supported by intranuclearpre-mRNA transport and metabolismthroughoutmetazos. NIH-HD1 8066 and NSF-BIR 9200027. Monday. Ribonucleoproteins and RNA Processing (1157-1162) 199a

1157 1158 THE EFFECT OF ADENOVIRUS INFECTION ON THE ORGANIZATION AND MICROINJECTION OF OLIGONUCLEOTIDES THAT NHIBIT PRE- TRANSPORT OF CELLULAR mRNA. ((S.S. Kanj, J.R. Nevins and L.I. Davis)) mRNA SPUCING IN VITRO RESULTS IN A REORGANIZATION Howard Hughes Medical Institute, Section of Genetics. Duke University Medical Center, Durham, NC 27710. OF SPUCING FACTORS IN VIVO. ((R. T. O'Keefe, A. Mayeda, A. R. Krainer, and D. L. Spector)) Cold Spring Harbor Laboratory, Cold We are using adenovirus (Ad) infection to explore the process of mRNA localization and Spring Harbor, NY 11724 transport to the cytoplasm. Late in infection of HeLa cells, Ad messages are effidently transported to the cytoplasm, whereas transport of cellularmessages is inhibited. This Factors in are a effect is dependent on two early viral proteins, ElB 55 kd and E4 ORF6. Using in situ involved pre-mRNA splicing organized in speckled analysis, we have localized cellular messages and splicing factors througbout the course of distribution within the mammalian cell nudeus. To assess the effect infection with wild type and mutant virus. Poly(A) RNA localization was profoundly of inhibition of pre-mRNA splicing on the organization of splicing altered during the early phase of infection with wild type Ad. Nucear transcript factors in vivo, oligonucleotides or antibodies that inhibit splicing in domains" were no longer apparent, and staining became very promninent at the nuckar were rim. Anti-Sm antibody also showed loss of intranuclear staining and pronounced vitro microinjected into HeLa cells. These probes caused a localization at the nuclear periphery. In addition, there was strong cytoplasmic staining. reorganization of splicing factors within the nudeus. Interchromatin The increase in cytoplasmic snRNPs was confrnmed by Western blotting with fractionated granule clusters decreased in number, became uniform in shape, and cells. In contrast to poly(A) RNA and snRNPs, SC-35 localization did not change at increased in both size and intensity of fluorescent labeling of splicing early times. These results suggest that Ad infecdon causes the release of mRNA-snRNPs from their association with SC-35 and the nuclear matrix early in infection. None of factors. These changes were transient and the organization of splicing these effects was seen when an Ad mutant deleted for ElB 55K was used. Infection with factors returned to their normal distribution by 24 hrs. following tsl25, whicb permits early but not late gene expression, produced effects identical to wild microinjection. However, nonspecific control oligonucleotides or type arguing that reorganization an process, rather than a passive result of is active antibodies did not affect the organization of splicing factors. production of large amounts of late viral transcripts. Late in infection, poly(A) The containing foci returned, but formed two distinct populations. Those that contained Ad oligonudeotides and antibodies that reorganized splicing factors also transcripts colocalized with snRNPs and SC-35. Another setdid nothybridize with Ad resulted in a reduction of transcription in vivo but not in vitro. This probes or show staining with SC-35, but did colocalize with snRNPs. The latter foci suggests that pre-mRNA splicing and transcription may be linked in were very distinct and localized at the nuclear periphery. They were not apparent in cells infected with the ElB deletion mutant. We propose that theserepresent sites of vivo. We believe that the enlarged interchromatin granule dusters we untransported cellular messages. Our results suggest that Ad infection causes a global observed may arise from the movement of splicing factors to their reorganization of cellulartranscript domains; during the early phase cellular foci are sites of storage and/or assembly. We propose that this reorganization disrupted, setting the stage for reorganization around the viral chromatin late in infection. of splicing factors may result from the inhibition of splicing in vivo. They further imply link between the structural organization of transcript domains within the nucleus and transport of messages to the cytoplasm.

1159 1160 RNA SYNTHESIS AND TRANSCRIPTION-RELATED ANTIGENS IN MAMMALIAN cDNAS HOMOLOGOUS TO YEAST RNA SPLICING ADENOVIRUS-INFECTED HeLa CELLS. Chen and N. Chaly)) FACTORS OF THE PUTATIVE RNA HELICASE/DEAH BOX FAMILY. ((X. EJ Department of Biology, Carleton University, Ottawa, Ont. KIS 5B6, ((SL Gee, Miller, K Aoyagi, and JG Conboy)) Life Sciences Division, Canada. Lawrence Berkeley Laboratory, Univ. of California, Berkeley, CA Among the"DEAD/H box" gene superfamily of putative RNA helicases are at A signal feature ofIytic adenovirus (Ad) infection is the assembly of least five pre-mRNA processing (PRP) proteins inS. cerevisiae which function several types of morphologically distinct nuclear inclusions (X. Chen in RNA splicing. Here we report isolation of three mammalian cDNAs with and N. Chaly, J. Cell Biol. -j,% 315a, 1991). In this study, we high homology to the yeast DEAH splicing factors PRP2, PRP16, PRP22 and examined the organization of Ad transcription in relation to the JAI. Using RT/PCR techniques with degenerate primers located at conserved peptide motifs, three partial clones (mDEAH6, and inclusions. EM autoradiography showed that RNA was synthesized mDEAH9, mDEAH47) were initially obtained from mouseerythroleukemia (MEL) cell cDNA. The primarily at the interface between the largestinclusions, the Clear amino acid sequences deduced from subsequently isolated longerMEL cDNAs Fibrillar Bodies (CFBs), and the peri-CFB nucleoplasmic region. After reveal that all three appear to have a similar tripartite structure (central domain long3H-uridine pulsetimes, label was more conspicuous in distal homologous to all DEAD/H proteins; C-terminus related DEAH proteins PRP2, peri-CFB regions, and both Very Dense Fibrillar Bodies (VDFBs) and 16, and 22; unique N-temiinus).mDEAH6 is -60% to both PRP16 and PRP22 the CFB-associated Dense Fibrillar Bodies (DFBs) acquired label. EM and may represent a functional homolog of one of these splicing factors. mDEAH9 is highly homologous to the yeast JAl/PRP40 gene (>65% identity immunogold labelling of transcription-related antigens showed that over 600 residues, excluding the N-terminus), also implicated in pre-mRNA the snRNP Sm antigen was concentrated in the distal peri-CFB region splicing in yeast. mDEAH47 exhibits slightly less homology (-50% identity) to and was also associated with DFBs and VDFBs, and that the Pl1 anti- the yeast splicing factors. All three probes hybridize on genomic "zooblots" gen showed a similar localization. On the other hand, DNA Topoiso- with DNA from a variety of mammalian species, as well as chicken DNA, merase1, implicated in both Ad DNA replication and transcription, implying a high degree of evolutionary conservation. Future experiments will mammalian was detected in CFBs, as well as in the peri-CFB, DFBs and VDFBs. test whether these genes can functionally complement yeast splicing mutants. Our results predict that the spliceosomal machinery which These results suggest that the various steps in the production of mediates ATP-dependent conformational changes in RNA conformation has been very mature are spatially segregated, with DNA transcribed Ad mRNA highly conservedthrough evolution from yeastto mammals. In addition, it is mainly in association with the surface of the CFBs, apd Ad RNA then becoming clear that mammals, like yeast, express a diverse array of DEAD/H spliced in the peri-CFB, and, perhaps, undergoing further maturation box proteins which are likely to play critical roles in regulating RNA in association with DFBs and VDFBs. (Supported by NSERCC) metabolism and function.

1161 1162

ISOLATION AND CHARACTERIZATION OF AN EUKARYOTIC CHARACTERIZATION OF SCHIZOSACCHAROMYCESPOMBE MRP MESSENGER RIBONUCLEASE. ((R.E. Dompenciel, V. Garnepudi and RIBONUCLEOPROTEN RNASE ANDINITAL INVESTIGATION NTO TIE RELATIONSHIP BETWEEN RNASE MRP AND RNASE P. ((l. Paluh and DA. D.R. Schoenberg)) Department of Pharmacology, Uniformed Services University Clayton)) Departmnt of Developmental Biology, Stanford University School of of the Health Sciences, Bethesda, MD 20814. Medicine, Stanford, CA 94305-5427. Twoessentil endoribonuclease ribonucloprteins (RNPs), RNase P and RNase MRP, The regulation of mRNA stability is a major control mechanism with a are lcalzed to bothmitochondial and nucar comparnentsin eukyotc cells RNase significant impact on gene expression. While sequences that impart instability or P has been chacterized motextensively in prodsts for its abilty to process pecursor seveas cleavage sites have been indentified in a number of mRNAs, little is tRNA moleculs to yied the maturetRNA 5 end. RNase MRP process RNA an p from to produce termini knownabout the enzymes that catalyze regulated mRNA decay. Recent work in for DNA at In the nucles,RNae MRPhas at last oneessentul nuclea Xesaaoiguslg has identifiod a protein factor in polysomalfractions of estrogen function rquired for processing of rRNA. he meaoan RNPswe precipited induced liver tissue that may be responsible for the differential catabolism sanichiomericly by an antibody deivedfrom thesar ofpatients withcatain autoinmune disorder n. In addition, the RNA compnens of observed for serum-coding mRNAs. Preliminary data proves this activity to be (usualluy wckl each ofthesetuique RNPsare sima in size(6 to400nts) and predicted to fold intosimilar anendonuclease with defined properties. The enzymatic activity generates a 'cage' stuc We are using the fision yeast, Schizosaccharovces pombe, order to investigate characteristic degradationfragment of 194 nucleotides upon digestion of a full- morclosely the between RNase MRP and RNase P. S. pombe gusellyiSan relationship is fro S. crewac vt psapsmore typical of meazan. length or5' end albumin transcript, which is independent of the presence or Imply,theRNAcomoentsipoih oftha RNPs are encoded by single nuclear seneinthi ye absence of Mg5", ATP or a5' cap structure.It is not inhibited by placental pxiAt.. asimpierleti semfr these investigations.Tegn S ribonuclease or N-ethylmaleimide, and is fairly stable and active over a range of for theRNAcomponent of pombc RNaaeMRP wascloned using a moue/Xenopur consensus and ecodait an RNA of 400 nts with predicteda pH from 6.6 to g.g. The messenger RNase is isolated through DEAE, Mono Q, co sucturesimilr tothat ofother known RNase MRP RNAs An RNA MonoS, phosphocellulose and preparativeisoelectric focusing resulting in an pcessingacdvity has beenisolatedthat isabletOproces Saccharomyces ori5 and activefraction containing a major 50 kDa polypeptide. Proof forits identity is humn D-loop mitochndrial RNA substes, asexpected forRNas MRP. Thisactivity in an in cm obtained by reconstitution ofits charactristic activity from protein separated on inhibited by iguc dethat plem entary to the highly conserved cage region of the S.pomreRNase MRP RNA. A seriesoftartia1 deletion derivatives of the 2D gels. Information gathered fromdetailed analysis on the mechanisms of RNaseMRPRN A genehave been mad and are being induced into cells. A deleion that induction and the nature of its interaction with suboeliular components will sig aificandy thesrucX ture of the RNA in expected to belethl. Chimeric provide a better understanding of the of g hormonlcontrol mRNA metaboliAm. RNA constructsand mutagn of the RNase MRP RNA genewill also be used idonto tifyshared uniqu factors required for the nuclear functons ofthce RNPs as well as to cidatfes ofsevral poeal regulatory stepsin mit bgbo ndtR NA poceing at th level of nucir tr nscaiption, RNP ass mbly, genat nport in the and imp into the mitochndri. 200a Ribonucleoproteins and RNA Processing (1163-1167). Monday

1163 1164 FUNCTIONAL ANALYSIS OF NAB2, A YEAST NUCLEAR COLOCALIZATION OF NUCLEAR POLYADENYLATED RNA-BINDING POLYADENYLATED RNA-BINDING PROTEIN. ((.T.Anderson and PROTEINS IN SACCHAROMYCES CEREVISIAE. ((S.M. Wilsonl, A.M. M.S.Swanson)) Department of Immunology & Medical Microbiology and Oberdorfl, M.R. Paddy2, and M.S. Swansonl)) lDepartment of Immunology Center for Mammalian Genetics. University of Florida, College of Medicine, & Medical Microbiology and Center for Mammalian Genetics, University of Gainesville, FL 32610-0266. Florida, College of Medicine, Gainesville, FL 32610-0266;2Center for Structural Biology and Department of Anatomy & Cell Biology, University of Although a variety of RNA-binding proteins bind directly to pre-mRNAs Florida, College of Medicine, Gainesville, FL 32610-0235. (Spon. by A. within the nucleus following transcriptional initiation, very litde is known about Lewin) the specific functions of this class of nuclear ribonucleoprotein. We have recently isolated and characterized nuclear polyadenylated RNA-kinding, or Pre-mRNA processing within the nucleus requires the coordinated assembly of NAB, proteins from Sacchsaromyces cerevisiae, and used the powerful genetic numerous pre-mRNA-binding proteins and snRNPs. To study the various system available in this yeast to geneate NAB conditional lethal alleles. NAB2 nuclear functions of pre-mRNA-binding proteins, we have recendy described is one of the major proteins directly associated with poly(A)+ RNA in vivo, and the isolation of nuclear polya,denylated RNA-hinding (NAB) proteins from is distributed witiin the nucleus in a distinct non-nucleolarpattern. he NAB2 Saccharomyces cerevisiae. NABI, NAB2, and NAB3 were isolated by a protein is composed of two separate domains that independenty bind RNA in method which utilizes UV light to crosslink proteins intimately associated with vitro: an arginine/glycine rich region (RGG box or GAR domain) that binds poly(A)+ RNA in vivo. NAB1 and NAB2 have been previously described and poly(G) and poly(U) and seven repeats of a C3H motif that bind poly(G), are structally related to human hnRNPs. The deduced amino acid sequence of poly(A), and poly(U), as well as single-sunded DNA. NAB2 also contains a NAB3 contains a single ribonucleoprotein consensus sequence RNA-binding glutanine/proline-rich tetrapeptide repeat motif, QQQP, which is probably domain most closely related to nmtan hnRNP Cl/C2 proteins, a glutamine- important for protein-protein inteactions. The length of this te de peat rich carboxyl teiminus and an acidic amino terminus related to nucleolin. Like is strain-dependent varying from two to nine copies. The NAB2 gene is NAB1 and NAB2, NAB3 is essential for cell viability. An analysis of the situated on chromosome Vfl between two other ribonucleoprotein genes, intranuclear distribution of the NAB proteins was investigated by cellular SUP44, the gene for the small ribosomal subunit S4, and CABI, which immunofluorescence combined with digital image processing. The NAB encodes a protein related to RNA-dependent NTPases. The NAB2 gene is proteins are pedominatly localized within the nucleoplasm in a non-nucleolar essential for cell growth. Using PCR-based random mutagenesis to generate and non-uniform pattern. In addition, the dtree-dimensional distribution of the mutations in the NAB2 gene, we have studied the role of NAB2 in pre-mRNA NAB proteins has been analyzed together with the intra-nucleolar distribution processing. of NOPI, a major nucleolar protein in yeast. This analysis indicates that the nucleolus forms a crescent-shaped structure which is interwoven with nucleoplasmic protrusions suggesting a possible mechanism for coordination of nuclear and nucleolar functions in yeast.

1165 1166 EPITOPE-TAGGED XENOPUS hnRNP Al LOCALIZES TO THE PHOSPHORYLATION OF THE HNRNP Al BY PROTEIN KJNASE C INHIBITS LAMPBRUSH CHROMOSOME LOOPS AND B AND C SNURPOSOMES STRAND ANNEALING. ((H. Idrissi, A. Kumar', Z. Damuni2, H. Guo2, and S. H. OF NOTOPHTHALMUS. Wilson'.)) 'Sealy Center for Molecular Science. The University of Texas [IT.C. Ingledue and B.K. Kayl] Curriculum in Genetics, University of North Medical Branch, Galveston, Texas 77555. Carolina at Chapel Hill, Chapel Hill, NC 27599. 2Department of Biological Sciences The University of South Carolina, Columbia, South Cwolina 29208. Currently, the cellular processes that control the progression of hnRNA to cytoplasmic mRNA are poorly understood. Heterogenous ribonucleoproteins (hnRNPs) are known to play an important role in these Phosphorylation regulates activity of several DNA binding proteins and is events which include splicing and transport hnRNP complexes form on implicated in regulating gene transcription, among other processes in the nascent RNA transcripts and are composed of more than twenty different cell. Phosphorylation of proteins in the heterogeneous ribonucleoprotein proteins. Antibodies to hnRNPs have been shown to bind to amphibian oomplex (hnRNP), including protein Al, has been suggested as a regulatory lampbrush chromosome loops which are sites of active transcription. Our step in pre-mRNA splicing. We examined the ability of the hnRNP Al to act laboratory has cloned two isoforms of Xenopus hnRNP Al, one of the most as a substrate for a number of serine/threonine protein kinases in vito. A prevalent hnRNPs. These isoforms differ by a 73 nucleotide deletion in the survey with seven protein kinases showed that hnRNP Al was domain cDNA. A polyclonal antibody generated against the C-terminal phosphorylated by casein kinase II, protamine kinase, protein kinase A and indicates two bands of 38 and 39 kDa on Western blots of oocyte extracts. protein kinase C (PKC). Autophosphorylation activated protein kinase and on chromosome of Immunofluorescent studies lampbrush spreads two forms of basic kinase failed to Al. Notophthalmus germinal vesicles using this antibody indicate that Al is myelin protein phosphorylate localized to the lampbrush chromosome loops and the B and C snurposomes. Proteolyis with trypsin and S.aureus V8 protease showed that PKC To distinquish between Al generated by oocyte injection of RNA and phosphorylated Al at three main sites, one in the UPI or N-terminal domain endogenous Al already present in the oocyte, the two isoforms have been and two in the C-terminal domain. Full and partial proteoWc cleavage epitope-tagged. Epitope-tagged Xenopus Al not only localizes to the followed by C-18 reverse phase chromatography and amino acid sequencing nucleus but also sub-localizes to the lampbrush chromosome loops and B revealed that Ser182, Ser192 and Ser199 were sites for PKC and C snurposomes of the newt Notophthdalus. in a pattern essendally phosphorylation. Phoephorylation of Al by PKC inhibited its strand similar to that generated by the polyclonal antibody to Al. Deletion annealing activity. Protein phosphatase 2A, but not protein phosphatasel analysis of these epitope-tagged constructs will help determine the dephosphorylated Al and reversed the inhbtory effect of PKC functional domains of hnRNPs . phosphorylation on strand annealing. The results are consistent with a role of phosphorylation of Al in regulating its strand annealing activity in vivo.

1167 SMALL NUCLEAR RNA ISOLATION PROM Bombyx mon ((F. dePreitas and Rj. Herrera)) Department of Biological Sciences, Florida Intemational University, Miami, Fla 33199

In order to perform functional studies involving isoforms of small nuclear RNAs, cloning of the transcript is necessary to get pennanent access tD the sequences that code for tese different RNA species. In ths study, methods of RNA purification are described. To achieve this goal, total RNA is isolated from nuclei of Bombyx mon cell lines and rapidly purified by high pressure ionic exchange liquid chromatography. This provides for good separation and enrichment for small nuclear RNAs from the higher molecular weight mRNA low molecular weight RNA and tRNA. This research was supported by U.S. Public Health Service Grant RR08205 to R.J.H.