The C. elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis

Tri Q. Nguyen 1 Hitoshi Sawa 0 Hideyuki Okano 0 John G. White ) 1 2 0 Division of Neuroanatomy, Osaka University Graduate School of Medicine , Suita, Osaka 565-0871 , Japan 1 Cellular Molecular Biology Program, Laboratory of Molecular Biology, University of Wisconsin , Madison, WI 53706 USA 2 Department of Anatomy, University of Wisconsin-Madison , WI 53706 USA postembryonic cytokineses and morphogenesis but have no essential SUMMARY Septins have been shown to play important roles in cytokinesis in diverse organisms ranging from yeast to mammals. In this study, we show that both the unc-59 and unc-61 loci encode Caenorhabditis elegans septins. Genomic database searches indicate that unc-59 and unc-61 are probably the only septin genes in the C. elegans genome. UNC-59 and UNC-61 localize to the leading edge of cleavage furrows and eventually reside at the midbody. Analysis of unc-59 and unc-61 mutants revealed that each septin requires the presence of the other for localization to the cytokinetic furrow. Surprisingly, unc-59 and unc-61 Cytokinesis is the act of cleaving a mother cell into two daughters and, as such, is one of the most fundamental of cellular processes. The basic process of cytokinesis consists of separating the cell constituents into two regions and pinching off and resealing the plasma membranes. There are two general strategies for cytokinesis in different organisms: progressive constriction of a contractile ring (Rappaport, 1996), or formation of a septum in the equatorial plane (Staehelin and Hepler, 1996). The contractile ring of animal cells is composed of actin filaments associated with many other proteins. The interaction of actin filaments and bipolar non-muscle myosin II generates a force that drives furrow ingression ultimately separating the cell into two independent entities (Cao and Wang, 1990; Satterwhite and Pollard, 1992; Sanders and Field, 1994). In contrast, although Saccharomyces cerevisiae has a contractile actomyosin ring (Lippincott and Li, 1998; Bi et al., 1998), it is not essential for cytokinesis; cells lacking F-actin can still divide (Ayscough et al., 1997). In S. cerevisiae, a bud emerges from the mother cell and enlarges whilst the mother cell maintains its volume. Vesicles fuse at the neck region to form new plasma membrane and the enlarging bud eventually separates from the mother cell by the deposition of chitin into mutants generally have normal embryonic development; however, defects were observed in post-embryonic development affecting the morphogenesis of the vulva, male tail, gonad, and sensory neurons. These defects can be at least partially attributed to failures in post-embryonic cytokineses although our data also suggest other possible roles for septins. unc-59 and unc-61 double mutants show similar defects to each of the single mutants. the mother-bud neck to form a new cell wall (reviewed by Sanders and Field, 1994; Longtine et al., 1996). Although these two processes of cytokinesis appear to be quite different, they both require a family of proteins called septins. The septin genes, CDC3, CDC10, CDC11 and CDC12, were first identified in S. cerevisiae for conferring defects in cytokinesis when mutated (Hartwell, 1971). The four gene products may form the highly ordered ~10 nm diameter neck filaments organized in a ring-like structure at the mother bud junction as previously described (Byers and Goetsch, 1976a) as these filaments are not seen in septin mutants (Byers and Groetsch, 1976b) and septins have been shown to form filaments in vitro (Field et al., 1996; Frazier et al., 1998). By immunofluorescence, septins first appear at the onset of G1 and persist for some time after cytokinesis. Temperature sensitive mutants of CDC3, CDC10, CDC11 and CDC12 cannot execute cytokinesis at the restrictive temperature and fail to localize septins at the mother bud neck. There is also a concomitant mislocalization of chitin deposition and failure to assemble a contractile ring (Haarer and Pringle, 1987; Kim et al., 1991; Ford and Pringle, 1991; DeMarini et al., 1997; Bi et al., 1998). Septins have been found in several other organisms since they were first identified in S. cerevisiae such as the yeast Candida albicans (DiDomenico et al., 1994) and the fungus Aspergillus nidulans (Momany and Hamer, 1997). The first animal septin gene, peanut (pnut), was identified and cloned in Drosophila. pnut mutants die after pupation due to cytokinesis failures in several larval tissues resulting in multinucleate cells. Pnut protein localizes to the leading edge of the cleavage furrow of dividing cells and resides in the midbody (Neufeld and Rubin, 1994). Since this discovery, septins have been identified in mammals (Nottenburg et al., 1990; Kato, 1990; Nakatsuru et al., 1994; Kinoshita et al., 1997; Hsu et al., 1998). Injection of antibody raised against a murine septin, Nedd5, resulted in cleavage furrow regression in SiHa cells (Kinoshita et al., 1997), suggesting that septins play a key role in cytokinesis of animal cells. The predicted septin proteins are at least 26% conserved in amino acid sequence over their entire length across species (reviewed by Longtine et al., 1996). At the structural level, all septins contain a putative P loop nucleoside triphosphatebinding motif at the N terminus of the protein. Most septins have a C terminus coiled-coil domain that has been generally thought to facilitate protein-protein interactions (Saraste et al., 1990; reviewed by Sanders and Field, 1994; Longtine et al., 1996). The P-loop has been speculated to play a role in regulating filament assembly. Field et al. (1996) showed that Drosophila Peanut, Sep-1 and Sep-2 have GTPase activity; these proteins form hetero-meric filaments of 7-9 nm diameter and ~26 nm length in vitro. Subsequently, Frazier et al. (1998) performed similar experiments in yeast showing that septins form filaments 7-9 nm in diameter with the majority of the filaments being 32 nm in length. However, it is not known whether filament assembly is necessary for septin function. Indeed, CDC10D mutants of S. cerevisiae do not form neck filaments or septin filaments in in vitro extracts, yet can localize proteins in the mother/bud neck and undergo cytokinesis (Frazier et al., 1998). Further support for the notion that septins have GTPase activity comes from other studies (Kinoshita et al., 1997) on Nedd5. In cells microinjected with GTPg S, septin filaments failed to assemble. Furthermore, they showed that Nedd5-GFP fusion proteins lacking the GTP binding domain interfered with the filamentous distribution of Nedd5. In this paper, we present molecular, immunolocalization, and phenotypic analyses of unc-59 (e261 and e1005) and unc-61 (e228 and n3169). We show that the unc-59 and unc61 loci encode two C. elegans septins. Both unc-59 and unc61 mutants show normal cytokinesis during embyrogenesis, yet exhibit failures in postembryonic cytokinesis. Double mutant analysis and (...truncated)