Abstract. We describe the phenotypic and molecular characterization of twinstar (tsr), an essential gene in Drosophila melanogaster. Two P-element induced alleles of tsr (tsr l and tsr 2) result in late larval or pupal lethality. Cytological examination of actively dividing tissues in these mutants reveals defects in cytokinesis in both mitotic (larval neuroblast) and meiotic (larval testis) cells. In addition, mutant spermatocytes show defects in aster migration and separation during prophase/prometaphase of both meiotic divisions. We have cloned the gene affected by these mutations and shown that it codes for a 17-kD protein in the cofilin/ ADF family of small actin severing proteins. A cDNA for this gene has previously been described by Edwards et al. (1994). Northern analysis shows that the tsr gene is expressed throughout development, and that the tsr a and tsr 2 alleles are hypomorphs that accumulate decreased levels of tsr mRNA. These findings prompted us to examine actin behavior during male meiosis to visualize the effects of decreased twinstar protein activity on actin dynamics in vivo. Strikingly, both mutants exhibit abnormal accumulations of F-actin. Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes. Later, during ana/telophase of both meiotic divisions, aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase, in contrast with wild-type. We discuss these resuits in terms of possible roles of the actin-based cytoskeleton in centrosome movement and in cytokinesis.ECULATED interactions between actin and actin binding proteins provide an important structural basis for many cellular processes in eukaryotes, such as cell-cell adhesion, cell outgrowth and motility, muscle contraction, and cytokinesis. Changes in the intracellular localization and organization of actin are mediated by a repertoire of actin-associated proteins. These fall into several classes based on their effects on actin assembly and dynamics in vitro: proteins with cross-linking, bundling, capping, severing, polymerizing/depolymerizing, monomer sequestering, and motor activities have been described (reviewed by Vandekerckove and Vancompernalle, 1992). However, defining the role that each actin-associated protein plays in vivo and how multiple activities are coordinated remains a challenging task.Proteins in the cofilin/actin depolymerizing factor (ADF) 1 family have been identified in a wide range of eukaryotic organisms, from yeast to plants to humans. These small molecular mass (15-20 kD) actin binding proteins consti-

Abstract. We describe here a new member of the kinesin superfamily in Drosophila, KLP3A . The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

We analyzed male meiosis in mutants of the chickadee (chic) locus, a Drosophila melanogaster gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. During ana-telophase of both meiotic divisions, they exhibit a central spindle less dense than wild type; certain chic allelic combinations cause almost complete disappearance of the central spindle. Moreover, chic mutant spermatocytes fail to form an actomyosin contractile ring. To further investigate the relationships between the central spindle and the contractile ring, we examined meiosis in the cytokinesis-defective mutants KLP3A and diaphanous and in testes treated with cytochalasin B. In all cases, we found that the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: When one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, we observed another consequence of chic mutations: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus, a gene that encodes a Drosophila member of the cofilin/ADF family of actin-severing proteins. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration.

and Smc3 proteins (reviewed in [7]), but other important proteins such as Pds5

The Zeste-White 10 (ZW10) and Rough Deal (ROD) proteins are part of a complex necessary for accurate chromosome segregation. This complex recruits cytoplasmic dynein to the kinetochore and participates in the spindle checkpoint. We used immunoaffinity chromatography and mass spectroscopy to identify the Drosophila proteins in this complex. We found that the complex contains an additional protein we name Zwilch. Zwilch localizes to kinetochores and kinetochore microtubules in a manner identical to ZW10 and ROD. We have also isolated a zwilch mutant, which exhibits the same mitotic phenotypes associated with zw10 and rod mutations: lagging chromosomes at anaphase and precocious sister chromatid separation upon activation of the spindle checkpoint. Zwilch's role within the context of this complex is evolutionarily conserved. The human Zwilch protein (hZwilch) coimmunoprecipitates with hZW10 and hROD from HeLa cell extracts and localizes to the kinetochores at prometaphase. Finally, we discuss immunoaffinity chromatography results that suggest the existence of a weak interaction between the ZW10/ ROD/Zwilch complex and the kinesin-like kinetochore component CENP-meta.