Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis.

Abstract

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-

p53-Mediated transactivation of LIMK2b links actin dynamics to cell cycle checkpoint control

“…This may not be totally unexpected because the ectopic expression of Xenopus LIMKs (Xlimk1/2) in oocytes inhibits meiotic progression (Takahashi et al, 2001). In addition, disruption of cofilin phosphorylation, which is normally transiently upregulated in the M-phase of the cell cycle (Amano et al, 2002), frequently results in failure of cytokinesis (Gunsalus et al, 1995;Abe et al, 1996;Hotulainen et al, 2005). These results suggest that proper modulation of cofilin phosphorylation is essential for successful cell division.…”

“…As LIMK2 phosphorylates and inactivates cofilin (Arber et al, 1998;Sumi et al, 1999), and the proper regulation of cofilin is required for cytokinesis (Gunsalus et al, 1995;Hotulainen et al, 2005), we then determined whether cofilin phosphorylation was deregulated in LIMK2b-GFP cells. As reported previously (Amano et al, 2002), phosphorylation of cofilin in control GFP cells increased in metaphase and then gradually decreased in telophase (Figure 4d).…”

The cofilin pathway in breast cancer invasion and metastasis

“…During Drosophila morphogenesis, the disruption of cofilin alleles by transposon mutagenesis caused an abnormal actin cytoskeleton characterized by large F-actin aggregates in primary spermatocytes and at the contractile ring during cytokinesis 92 , as well as the follicular epithelium during ovarian development 58 . Loss-of-function mutations in genes that regulate cofilin phosphorylation, such as the cofilin phosphatase slingshot and the cofilin kinase LIM kinase (LIMK), further support a role for cofilin in regulating actin dynamics during Drosophila morphogenesis.…”

TheDrosophila Cog5Homologue Is Required for Cytokinesis, Cell Elongation, and Assembly of Specialized Golgi Architecture during Spermatogenesis

“…In live squashes, DNA was visualized with 10 g/ml Hoechst 33342 dye. To visualize ␣-tubulin with F-actin, GFP-Fws with Lava Lamp, GFP-Fws with ␣-mannosidase, or endogenous Fws, cells were squashed and then fixed with 4% formaldehyde as described by Gunsalus et al (1995). In all cases, GFP-Fws was visualized directly by exciting the green fluorescent protein (GFP) moiety, even in fixed material.…”