Centromeric chromatin – spindle microtubule interactions mediated by kinetochores drive chromosome segregation. We have developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at < 5 nm accuracy — to elucidate the protein architecture of human metaphase kinetochores. Delta analysis, when correlated with tension states of spindle-attached sister kinetochore pairs, provided information on mechanical properties of protein linkages within kinetochores. Treatment with taxol—which suppresses microtubule dynamics, eliminates tension at kinetochores, and activates the spindle checkpoint—resulted in specific large-scale changes in kinetochore architecture. Cumulatively, Delta analysis revealed compliant linkages close to the centromeric chromatin, suggests a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and reveals architectural changes induced by taxol treatment. The methods described here should also be applicable to other intermediate-scale biological machines in cells.

RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.

We report the interactions amongst 20 proteins that specify their assembly to the centromere–kinetochore complex in human cells. Centromere protein (CENP)-A is at the top of a hierarchy that directs three major pathways, which are specified by CENP-C, -I, and Aurora B. Each pathway consists of branches that intersect to form nodes that may coordinate the assembly process. Complementary EM studies found that the formation of kinetochore trilaminar plates depends on the CENP-I/NUF2 branch, whereas CENP-C and Aurora B affect the size, shape, and structural integrity of the plates. We found that hMis12 is not constitutively localized at kinetochores, and that it is not essential for recruiting CENP-I. Our studies also revealed that kinetochores in HeLa cells contain an excess of CENP-A, of which ∼10% is sufficient to promote the assembly of normal levels of kinetochore proteins. We elaborate on a previous model that suggested kinetochores are assembled from repetitive modules (Zinkowski, R.P., J. Meyne, and B.R. Brinkley. 1991. J. Cell Biol. 113:1091–110).

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore. INTRODUCTIONThe mitotic checkpoint is a fail-safe mechanism that ensures accurate chromosome segregation by preventing cells from prematurely exiting mitosis in the presence of unaligned chromosomes (Nicklas, 1997;Rieder and Salmon, 1998;Amon, 1999). This checkpoint system is highly sensitive, because even a single unaligned chromosome is sufficient to block cells from entering anaphase (Rieder et al., 1994;Li and Nicklas, 1997). The mitotic checkpoint has been shown to monitor both microtubule attachment and tension generated across sister kinetochores by poleward forces (Rieder et al., 1994;Li and Nicklas, 1997;Waters et al., 1998). Failure of the mitotic checkpoint causes cells to exit mitosis in the presence of unaligned chromosomes and is a major mechanism responsible for aneuploidy (Jallepalli and Lengauer, 2001). Seven mitotic checkpoint genes, BUB1, BUB2, BUB3, MAD1, MAD2, MAD3, and MPS1, were originally identified via genetic screens in Saccharomyces cerevisiae (Hoyt et al., 1991;Li and Murray, 1991;Weiss and Winey, 1996). These genes act along two separate mitotic checkpoint pathways (Clarke and Gimenez-Abian, 2000;Daum et al., 2000;Gardner and Burke, 2000). MPS1, BUB1, BUB3, MAD1, MAD2, and MAD3 monitor kinetochore microtubule attachments and prevent premature chromosome segregation by inhibiting degradation of securin/Pds1 and mitotic cyclins (Wassmann and Benezra, 2001;Peters, 2002). BUB2 acts along a different pathway that monitors spindle integrity and orientation and prevents premature cytokinesis by inhibiting the degradation of the mitotic cyclin Clb2 (Alexandru et al., 1999;Fesquet et al., 1999;Fraschini et al., 1999;Li, 1999;Bardin et al., 2000;Bloecher et al., 2000;Pereira et al., 2000).Many of the mitotic checkpoint genes in yeast are evolutionarily conserved, because orthologues of M...

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for centromere cohesion (Kitajima et al., 2006). In this study, we show that hSgo2 exhibits a dynamic distribution pattern, and that its localization depends on the BUB1 and Aurora B kinases. hSgo2 is concentrated at the inner centromere of unattached kinetochores, but extends toward the kinetochores that are under tension. This localization pattern is reminiscent of MCAK, which is a microtubule depolymerase that is believed to be a key component of the error correction mechanism at kinetochores. Indeed, we found that hSgo2 is essential for MCAK to localize to the centromere. Delocalization of MCAK accounts for why cells depleted of hSgo2 exhibit kinetochore attachment defects that go uncorrected, despite a transient delay in the onset of anaphase. Consequently, these cells exhibit a high frequency of lagging chromosomes when they enter anaphase. We confirmed that hSgo2 is associated with PP2A, and we propose that it contributes to the spatial regulation of MCAK activity within inner centromere and kinetochore.