Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly

The functions of Beclin‐1 in macroautophagy, tumorigenesis and cytokinesis are thought to be mediated by its association with the PI3K‐III complex. Here, we describe a new role for Beclin‐1 in mitotic chromosome congression that is independent of the PI3K‐III complex and its role in autophagy. Beclin‐1 depletion in HeLa cells leads to a significant reduction of the outer kinetochore proteins CENP‐E, CENP‐F and ZW10, and, consequently, the cells present severe problems in chromosome congression. Beclin‐1 associates with kinetochore microtubules and forms discrete foci near the kinetochores of attached chromosomes. We show that Beclin‐1 interacts directly with Zwint‐1—a component of the KMN (KNL‐1/Mis12/Ndc80) complex—which is essential for kinetochore–microtubule interactions. This suggests that Beclin‐1 acts downstream of the KMN complex to influence the recruitment of outer kinetochore proteins and promotes accurate kinetochore anchoring to the spindle during mitosis.     

Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment

For proper chromosome segregation, all kinetochores must achieve bipolar microtubule （MT） attachment and subsequently align at the spindle equator before anaphase onset. The MT minus end-directed motor dynein/dynactin binds kinetoehores in prometaphase and has long been implicated in chromosome congression. Unfortunately, inactivation of dynein usually disturbs spindle organization, thus hampering evaluation of its kinetochore roles. Here we specifically eliminated kinetochore dynein/dynactin by RNAi-mediated depletion of ZW10, a protein essential for kinetochore localization of the motor. Time-lapse microscopy indicated markedly-reduced congression efficiency, though congressing chromosomes displayed similar velocities as in control cells. Moreover, cells frequently failed to achieve full chromosome alignment, despite their normal spindles. Confocal microcopy revealed that the misaligned kinetochores were monooriented or unattached and mostly lying outside the spindle, suggesting a difficulty to capture MTs from the opposite pole. Kinetoehores on monoastral spindles were dispersed farther away from the pole and exhibited only mild oscillation. Furthermore, inactivating dynein by other means generated similar phenotypes. Therefore, kinetochore dynein produces on monooriented kinetochores a poleward pulling force, which may contribute to efficient bipolar attachment by facilitating their proper microtubule captures to promote congression as well as full chromosome alignment.     

The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.     

Neural Wiskott-Aldrich syndrome protein is required for accurate chromosome congression and segregation

The accurate distribution and segregation of replicated chromosomes through mitosis is crucial for cellular viability and development of organisms. Kinetochores are responsible for the proper congression and segregation of chromosomes. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) localizes to and forms a complex with kinetochores in mitotic cells. Depletion of NWASP by RNA interference causes chromosome misalignment, prolonged mitosis, and abnormal chromosomal segregation, which is associated with decreased proliferation of N-WASP-deficient cells. N-WASP-deficient cells display defects in the kinetochores recruitment of inner and outer kinetochore components, CENP-A, CENP-E, and Mad2. Live-cell imaging analysis of GFP-α-tubulin revealed that depletion of N-WASP impairs microtubule attachment to chromosomes in mitotic cells. All these results indicate that N-WASP plays a role in efficient assembly of kinetochores and attachment of microtubules to chromosomes, which is essential for accurate chromosome congression and segregation.     

Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase

Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase.     

CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore‐microtubule dynamics to promote mitotic progression and fidelity

Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore–microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b–CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin–CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1–astrin–Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments.     

Annexin A2 is required for the early steps of cytokinesis

Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium- and phospholipid-binding protein implicated in cortical actin remodeling. We report that annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In the absence of annexin A2, the small GTPase RhoA—which regulates cortical cytoskeletal rearrangement—fails to form a compact ring at the equatorial plane. Furthermore, annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in central spindle–equatorial plasma membrane communication.     

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.     

SHCBP1 is required for midbody organization and cytokinesis completion

The centralspindlin complex, which is composed of MKLP1 and MgcRacGAP, is one of the crucial factors involved in cytokinesis initiation. Centralspindlin is localized at the middle of the central spindle during anaphase and then concentrates at the midbody to control abscission. A number of proteins that associate with centralspindlin have been identified. These associating factors regulate furrowing and abscission in coordination with centralspindlin. A recent study identified a novel centralspindlin partner, called Nessun Dorma, which is essential for germ cell cytokinesis in Drosophila melanogaster. SHCBP1 is a human ortholog of Nessun Dorma that associates with human centralspindlin. In this report, we analyzed the interaction of SHCBP1 with centralspindlin in detail and determined the regions that are required for the interaction. In addition, we demonstrate that the central region is necessary for the SHCBP1 dimerization. Both MgcRacGAP and MKLP1 are degraded once cells exit mitosis. Similarly, endogenous and exogenous SHCBP1 were degraded with mitosis progression. Interestingly, SHCBP1 expression was significantly reduced in the absence of centralspindlin, whereas centralspindlin expression was not affected by SHCBP1 knockdown. Finally, we demonstrate that SHCBP1 depletion promotes midbody structure disruption and inhibits abscission, a final stage of cytokinesis. Our study gives novel insight into the role of SHCBP in cytokinesis completion.     

Cancer-upregulated gene 2 (CUG2), a new component of centromere complex,is required for kinetochore function

We previously identified cancer-upregulated gene 2 (CUG2) as a commonly up-regulated gene in various human cancer tissues, especially in ovary, liver, and lung (Lee et al., 2007a). CUG2 was determined to be a nuclear protein that exhibited high proto-oncogenic activities when overexpressed in NIH3T3 mouse fibroblast cells. To identify other cellular functions of CUG2, we performed yeast two-hybrid screening and identified CENP-T, a component of CENP-A nucleosome complex in the centromere, as an interacting partner of CUG2. Moreover, CENP-A, the principle centromeric determinant, was also found in complex with CENP-T/CUG2. Immunofluorescent staining revealed the co-localization of CUG2 with human centromeric markers. Inhibition of CUG2 expression drastically affected cell viability by inducing aberrant cell division. We propose that CUG2 is a new component of the human centromeric complex that is required for proper chromosome segregation during mitosis.     

Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss

Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.     

Parallel pathways for recruiting effector proteins determine centromere drive and suppression

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Annexin 11 is required for midbody formation and completion of the terminal phase of cytokinesis

Annexins are Ca(2+)-binding, membrane-fusogenic proteins with diverse but poorly understood functions. Here, we show that during cell cycle progression annexin 11 translocates from the nucleus to the spindle poles in metaphase and to the spindle midzone in anaphase. Annexin 11 is recruited to the midbody in late telophase, where it forms part of the detergent-resistant matrix that also contains CHO1. To investigate the significance of these observations, we used RNA interference to deplete cells of annexin 11. A combination of confocal and video time-lapse microscopy revealed that cells lacking annexin 11 fail to establish a functional midbody. Instead, daughter cells remain connected by intercellular bridges that contain bundled microtubules and cytoplasmic organelles but exclude normal midbody components such as MKLP1 and Aurora B. Annexin 11-depleted cells failed to complete cytokinesis and died by apoptosis. These findings demonstrate an essential role for annexin 11 in the terminal phase of cytokinesis.     

Ran is required before metaphase for spindle assembly and chromosome alignment and after metaphase for chromosome segregation and spindle midbody organization

The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.     

The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus

In Caulobacter crescentus, the genes encoding the chromosome partitioning proteins, ParA and ParB, are essential. Depletion of ParB resulted in smooth filamentous cells in which DNA replication continued. Immunofluorescence microscopy revealed that the formation of FtsZ rings at the mid-cell, the earliest molecular event in the initiation of bacterial cell division, was blocked in cells lacking ParB. ParB binds sequences near the C. crescentus origin of replication. Cell cycle experiments show that the formation of bipolarly localized ParB foci, and presumably localization of the origin of replication to the cell poles, preceded the formation of FtsZ rings at the mid-cell by 20 min. These results suggest that one major function of ParA and ParB may be to regulate the initiation of cytokinesis in C. crescentus.     

Sgt1, a co‐chaperone of Hsp90 stabilizes Polo and is required for centrosome organization

Sgt1 was described previously in yeast and humans to be a Hsp90 co‐chaperone and required for kinetochore assembly. We have identified a mutant allele of Sgt1 in Drosophila and characterized its function. Mutations in sgt1 do not affect overall kinetochore assembly or spindle assembly checkpoint. sgt1 mutant cells enter less frequently into mitosis and arrest in a prometaphase‐like state. Mutations in sgt1 severely compromise the organization and function of the mitotic apparatus. In these cells, centrioles replicate but centrosomes fail to mature, and pericentriolar material components do not localize normally resulting in highly abnormal spindles. Interestingly, a similar phenotype was described previously in Hsp90 mutant cells and correlated with a decrease in Polo protein levels. In sgt1 mutant neuroblasts, we also observe a decrease in overall levels of Polo. Overexpression of the kinase results in a substantial rescue of the centrosome defects; most cells form normal bipolar spindles and progress through mitosis normally. Taken together, these findings suggest that Sgt1 is involved in the stabilization of Polo allowing normal centrosome maturation, entry and progression though mitosis.     

Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis‐segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.     

A dual role for Bub1 in the spindle checkpoint and chromosome congression

The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. In this study, we use live-cell imaging coupled with RNA interference to investigate the functions of human Bub1. We find that Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments.