Successful divisions of eukaryotic cells require accurate and coordinated cycles of

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. and correct alternation of the S and M phases of the cell cycle. and cell biological studies of mammalian tissue-culture cells. Meiotic and mitotic spindles have been shown to consist of microtubules CAPZA1 and associated proteins (for review, Desai and Mitchison 1997). The microtubules assemble by polymerization of -tubulin dimers. Polymerization is usually initiated at microtubule-organizing centers (MTOCs). Microtubules are polar; they contain minus TR-701 cost ends TR-701 cost that usually remain associated with the MTOC and plus ends that lengthen into the cytoplasm and undergo rapid growth and shrinkage. Motor proteins of the dynein and kinesin families use the polarity of these microtubules to generate pressure in either the plus or minus end direction (for review, Hoyt and Geiser 1996). The role of the MTOC in spindle formation is still unclear. MTOCs can vary greatly in morphology, as is obvious when comparing centrosomes in animal cells and spindle pole body in yeast (Stearns and Winey 1997). In addition, spindles can be created without centrosomes in certain cell divisions, for instance during female meiosis in many animals (McKim and Hawley 1995). However, in most cell divisions, the MTOCs duplicate, individual to reverse poles, and form two sites for microtubule nucleation. In this way, MTOCs contribute to the bipolar nature of the spindle, as well as to quick and directional assembly of microtubules. The mitotic functions of the spindle apparatus include separation of TR-701 cost the centrosomes, segregation of sister chromatids, and specification of the cleavage-plane position. These different tasks depend on correct localization and activation of a large number of microtubule-associated motor proteins. In addition, activity of these motor proteins needs to be coordinated with microtubule assembly and disassembly. Multiple levels of regulation control the timing and execution of these mitotic processes. The cyclin-dependent protein kinase Cdk1/Cdc2 is the important regulator of mitosis in all eukaryotes analyzed (for reviews, TR-701 cost Morgan 1997; Mendenhall and Hodge 1998). Activation of Cdk1/Cdc2 in association with a mitotic cyclin is essential for access into mitosis, whereas exit from mitosis requires inactivation of this kinase and degradation of the cyclins. Phosphorylation of substrates by Cdk1/Cdc2 is usually thought to induce major events in M phase, such as chromosome condensation, nuclear envelope degradation, and spindle formation. Checkpoint controls can interrupt the periodic activation and inactivation of Cdk’s, thereby pausing cell cycle progression and allowing time for the completion of earlier events (for evaluate, Elledge 1996). It is likely that components with important TR-701 cost functions in the accurate segregation of chromosomes are yet to be recognized. In addition, it remains largely unknown how the many molecules involved are temporally and spatially regulated. The nematode provides an animal model excellently suited for further cell division studies. The transparency of allows monitoring of cell division and chromosome segregation in living animals. The invariance of the cell lineage has allowed a precise description of the time and plane of division for every somatic cell (Sulston and Horvitz 1977; Sulston et al. 1983), which provides a unique tool in the identification and characterization of cell cycle mutants. The cloning of genes defined by such mutants is usually facilitated by powerful genetics (Brenner 1974) and a completely sequenced genome (embryogenesis have been described in detail (examined by Strome 1993; White and Strome 1996). To improve our understanding of the mechanisms involved in accurate chromosome segregation in animal cells, we have characterized the gene mutants cycle through abortive mitoses, alternated with subsequent rounds of DNA replication (Albertson et al. 1978; Sulston and Horvitz 1981). We have cloned the gene and found that it encodes a novel protein localized most prominently at the centrosomes during mitosis and at the spindle in meiosis. The defects observed in mutants and the localization of the LIN-5 protein indicate primary functions in chromosome segregation and spindle positioning and potential secondary functions in cytokinesis and coupling the S and M phases. Materials and Methods Strains and Genetics strains were derived from the wild-type Bristol strain N2 and Bergerac strain RW7000 and cultured using standard techniques as.