Monday. Dynein and Kinesin 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 Cell 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 microtubule 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 dyneins (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 protein 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<pf2<sup-pf-3<wt). (2) These defects are more pronounced Analogous to their effects on sliding. inhibitors of calmodulin and PKA decrease in vitro in distal/medial cross-sections than in proximal cross-sections. (3) Analysis offlagellar incorporation of phosphate in pJ14 axonemes but not in wild type axonemes. Together extracts by FPLC and SDS-PAGE indicate that a specific dynein heavy chain is these results suggest that in Chlainydopnonas flagella dynein activity is regulated by missing in pf3, but not in pf2 and sup-pf-3. (4) When viewed in longitudinal section, phosphorylation, and the radial spokes may modulate this mechanism. We are currently all three DRC mutants lack a crescent shaped density beneath the second radial identifying substrates involved in dynein regulation. spoke. We propose that this structure corresponds in part to the location of the DRC. This position is appropriate for a complex that is thought to mediate signals between the inner dynein arms and radial spokes. (Supported by grants from the NSF and MOD to MEP and from NIH to the Boulder Laboratory for 3-D Fine Structure). 938 939 A 29 kDa cAMP-DEPENDENT PHOSPHOPROT'EIN IS DIRECTLY SEA URCHIN SPERM 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 proteins 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 microtubules 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 oocyte 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 dynactin (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.