Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

Abstract

Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions. INTRODUCTIONMitotic spindle formation is critical for proper chromosome congression, alignment, and segregation during cell division (Compton, 2000;Scholey et al., 2003;Kline-Smith and Walczak, 2004). Microtubules (MTs) undergo drastic changes in their dynamics and organization as the cell enters mitosis to form the bipolar spindle apparatus. As the nuclear membrane breaks down at the G2/M transition, interphase cytoskeletal MTs are destabilized, and mitotic spindle MTs nucleate from two opposing centrosomes/spindle poles with increased growth and turnover rates (Saxton et al., 1984;Zhai et al., 1996). This mitosis-specific change of microtubule behavior indicates the presence of cell cycle-specific regulators of spindles. Indeed, studies identified many important microtubule-associated proteins (MAPs) that regulate different aspects of mitotic spindle dynamics, such as the efficiency of MT nucleation and minus-end focusing at spindle poles, MT plus-and minus-end dynamics, and overall spindle stability (Kline-Smith and Walczak, 2004). However, the intricate regulatory mechanisms for spindle organization are not completely understood, and there are most likely additional factors that contribute to proper spindle regulation during mitosis.Cohesin is a conserved and essential multiprotein complex required for sister chromatid cohesion (Losada et al., 1998;Uhlmann and Nasmyth, 1998;Toth et al., 1999). Genetic studies in both yeast and metazoans revealed that inhibition of cohesin function leads to premature separation of sister chromatids, chromosome misalignment, and meloteric attachment of spindles to...

Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair

“…Similar results were obtained with additional lentivirus-transduced short hairpin RNAs (shRNAs) against SA1 and SA2 (data not shown). Mitotic indices were comparable in synchronized control and SA1-and SA2-depleted cells, and both proteins were depleted equally efficiently in metaphase cells without a prominent cohesion defect as previously described (40), indicating that the result is not due to the different effects of SA1 and SA2 depletion on cell cycle progression and/or selection of undepleted cells in mitosis (see Fig. S7 in the supplemental material).…”

“…HeLa cells were transfected twice (except for Rad21 small interfering RNA [siRNA], which was once) 24 h apart with siRNAs at a final concentration of 5 nM using HiPerFect transfection reagent according to the manufacturer's instructions (Qiagen). siRNAs directed against hSMC1 (5=-CACCATCACACTTTAATTCCA-3=), hRad21 (5=-A TCGATGAGCCCATTATTGAA-3=), SA1 (5=-CACGTAGAATCAGATG TTCTA-3=), and SA2 (5=-TCGGTGGTAGATGATTGGATA-3=) as described previously (40) and NIPBL (5=-CTAGCTGACTCTGACAATAA A-3=) or a negative-control siRNA (Qiagen) were used. Cells were harvested for Western blot analyses or subjected to laser microirradiation, approximately 24 h after the final transfection.…”

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…Interestingly, cytoplasmic SMC1 protein of the cohesin complex, was found to localise to centrosomes (Gregson et al, 2001;Guan et al, 2008;Kong et al, 2009). In vertebrates a cohesin function was shown to be necessary in aster assembly and required in microtubule spindle organisation (Kong et al, 2009).…”

“…In vertebrates a cohesin function was shown to be necessary in aster assembly and required in microtubule spindle organisation (Kong et al, 2009). Cohesin has been shown to interact with NuMa prior to mitosis, it is hypothesized that NuMa transports cohesin to spindle poles in a dynein dependent manner (Gregson et al, 2001;Kong et al, 2009;Merdes et al, 2000).…”

“…Since asters formed normally in XCEP63 depleted extracts, this suggests that XCEP63 function does not relate specifically to microtubule polymerisation or to an integral centrosome structure component. It has been hypothesised that cohesin functions in spindle assembly through SMC domain dependent bundling and stabilisation of fibres, albeit microtubules instead of chromatin fibres (Kong et al, 2009). The probable functional importance of the SMC domain within XCEP63 is supported by the fact that it is maintained across vertebrate species.…”