Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.

An integral part of cell division is the separation of daughter cells via cytokinesis.There is now good evidence that the completion of cytokinesis requires coordinated membrane trafficking to deliver new membrane to the tip of the furrow and to complete the abscission. Here we have examined membrane traffic in cytokinesis and describe several novel observations. First, we show that Rab11-and FIP3-containing recycling endosomes accumulate near the cleavage furrow and are required for successful completion of cytokinesis. Second, we demonstrate that the Rab11-FIP3 protein complex is intimately involved in the delivery of endosomes to the cleavage furrow. Significantly, although FIP3 recruitment to endosomes is Rab11 dependent, we find that the targeting of FIP3 to the midbody is independent of Rab11. Third, we show that the Rab11-FIP3 complex is required for a late stage of cytokinesis, possibly abscission. Finally, we demonstrate that localization of FIP3 is subject to substantial spatial and temporal regulation. These data provide the first detailed analysis of recycling endosomes in cell division and provide a new model for membrane traffic to the furrow. We propose that the dynamic Rab11-FIP3 interaction controls the delivery, targeting, and fusion of recycling endosomes with furrow during late cytokinesis and abscission. INTRODUCTIONAn integral part of cell division is the physical separation of two daughter cells via a process known as cytokinesis (Scholey et al., 2003). At least two distinct processes are required for successful cytokinesis: formation and constriction of an acto-myosin contractile ring and the delivery of new membrane to the progressing cleavage furrow (O'Halloran, 2000;Scholey et al., 2003). Both of these steps are tightly controlled and crucial for cell abscission, the final separation of the two cells. Although the function of the acto-myosin ring in cell division is well understood, we are only beginning to understand the role of membrane transport during cytokinesis. Evidence suggests that insertion of new membrane at the apex of cleavage furrow is crucial for the successful completion of cellularization in Drosophila embryos (Rothwell et al., 1999;Zhang et al., 2000). Similar requirements for membrane transport and fusion were also observed in Xenopus laevis eggs (Byers and Armstrong, 1986;Bieliavsky et al., 1992).The plasma membrane of the cleavage furrow is distinct in its lipid and protein composition from the rest of the plasma membrane (Emoto et al., 1996;Umeda and Emoto, 1999;Emoto and Umeda, 2000). The unique composition of cleavage furrow plasma membrane may underscore its ability to be deformed during ingression, as a cell is pinched in two, as well as possibly generating the signals that regulate progression of cytokinesis. Thus, in addition to the delivery of the membrane to compensate for the expanding plasma membrane surface, membrane traffic during cytokinesis could also mediate the delivery of proteins that control the ingression of the cleavage furrow as well as cell-...

Entry into anaphase and exit from mitosis depend on a ubiquitin-protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin-protein ligase complex SCFCdc4 required for the initiation of DNA replication.