No abstract

Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that γ-tubulin–dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.

SUMMARY Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but mechanisms determining spindle length are poorly understood. Based on a 2D model of meiotic spindle assembly, we predicted that higher localized microtubule (MT) depolymerization rates could generate the shorter spindles observed in egg extracts of X. tropicalis compared to X. laevis. We found that katanin-dependent MT severing was increased in X. tropicalis, which lacks an inhibitory phosphorylation site in the p60 catalytic subunit that is present in X. laevis p60. Katanin inhibition lengthened spindles in both species, and kinetochore fibers extended through X. tropicalis spindle poles disrupting them. In both X. tropicalis extracts and the spindle simulation, a threshold number of stable kinetochore fibers could overwhelm MT depolymerization, leading to similar phenotypes. Thus, mechanisms have evolved in different species to scale spindle size and coordinate regulation of multiple MT populations in order to generate a robust steady state structure.

In 1993, an enzyme with an ATP-dependent microtubule severing activity was purified from sea urchin eggs and named katanin, after the Japanese for sword. Now we know that katanin, spastin and fidgetin form a family of closely related microtubule severing enzymes that is widely distributed in eukaryotes ranging from Tetrahymena and Chlamydomonas to humans (Table 1, Figure 1A). Here we review the diverse in vivo functions of these proteins and the recent significant advances in deciphering the biophysical mechanism of microtubule severing.

In Saccharomyces cerevisiae, the HMR-E silencer blocks site-specific interactions between proteins and their recognition sequences in the vicinity of the silencer. Silencer function is correlated with the firing of an origin of replication at HMR-E. An essential gene with a role in transcriptional silencing was identified by means of a screen for mutations affecting expression of HMR. This gene, known as ORC2, was shown to encode a component of the origin recognition complex that binds yeast origins of replication. A temperature-sensitive mutation in ORC2 disrupted silencing in cells grown at the permissive temperature. At the restrictive temperature, the orc2-1 mutation caused cell cycle arrest at a point in the cell cycle indicative of blocks in DNA replication. The orc2-1 mutation also resulted in the enhanced mitotic loss of a plasmid, suggestive of a defect in replication. These results provide strong evidence for an in vivo role of ORC in both chromosomal replication and silencing, and provide a link between the mechanism of silencing and DNA replication.