钙调素（CaM）在中体上的分布及参与胞质分裂的调控

钙调素(CaM)是细胞内Ca^2 的主要受体,在细胞增殖、分化、凋亡、迁移等过程中都发挥着重要的调控作用。采用GFP标记技术,我们观察了GFP—CaM在胞质分裂期HeLa细胞中的动态分布,发现在胞质分裂后期,GFP—CaM与中体紧密相连。抑制CaM的活性会阻止中体的解聚。进一步观察发现,CaM与γ-微管蛋白共分布在中体两侧,抑制CaM活性也会引起中体γ-微管蛋白解离的延迟。本实验结果说明分布在中体上的CaM很可能通过影响中体微管的稳定,参与调控胞质分裂的完成。     

Functional involvement of human discs large tumor suppressor in cytokinesis

Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited a similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis.     

Kinesin-like protein CHO1 is required for the formation of midbody matrix and the completion of cytokinesis in mammalian cells

CHO1 is a mammalian kinesin-like motor protein of the MKLP1 subfamily. It associates with the spindle midzone during anaphase and concentrates to a midbody matrix during cytokinesis. CHO1 was originally implicated in karyokinesis, but the invertebrate homologues of CHO1 were shown to function in the midzone formation and cytokinesis. To analyze the role of the protein in mammalian cells, we mutated the ATP-binding site of CHO1 and expressed it in CHO cells. Mutant protein (CHO1F') was able to interact with microtubules via ATP-independent microtubule-binding site(s) but failed to accumulate at the midline of the central spindle and affected the localization of endogenous CHO1. Although the segregation of chromosomes, the bundling of midzone microtubules, and the initiation of cytokinesis proceeded normally in CHO1F'-expressing cells, the completion of cytokinesis was inhibited. Daughter cells were frequently entering interphase while connected by a microtubule-containing cytoplasmic bridge from which the dense midbody matrix was missing. Depletion of endogenous CHO1 via RNA-mediated interference also affected the formation of midbody matrix in dividing cells, caused the disorganization of midzone microtubules, and resulted in abortive cytokinesis. Thus, CHO1 may not be required for karyokinesis, but it is essential for the proper midzone/midbody formation and cytokinesis in mammalian cells.     

Adherens junctions determine the apical position of the midbody during follicular epithelial cell division

Cytokinesis is asymmetric along the apical–basal axis of epithelial cells, positioning the midbody near the apical domain. However, little is known about the mechanism and purpose of this asymmetry. We use live imaging of Drosophila follicle cell division to show that asymmetric cytokinesis does not result from intrinsic polarization of the main contractile ring components. We show that adherens junctions (AJs) maintain close contact with the apical side of the contractile ring during cytokinesis. Asymmetric distribution of AJ components within follicle cells and in the otherwise unpolarized S2 cells is sufficient to recruit the midbody, revealing that asymmetric cytokinesis is determined by apical AJs in the epithelia. We further show that ectopic midbody localization induces epithelial invaginations, shifting the position of the apical interface between daughter cells relative to the AB axis of the tissue. Thus, apical midbody localization is essential to maintain epithelial tissue architecture during proliferation.     

Syntaxin 2 and endobrevin are required for the terminal step of cytokinesis in mammalian cells

The terminal step of cytokinesis in animal cells is the abscission of the midbody, a cytoplasmic bridge that connects the two prospective daughter cells. Here we show that two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, specifically localize to the midbody during cytokinesis in mammalian cells. Inhibition of their function by overexpression of nonmembrane-anchored mutants causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission but not further upstream events, such as furrowing, are affected. These results indicate that successful completion of cytokinesis requires a SNARE-mediated membrane fusion event and that this requirement is distinct from exocytic events that may be involved in prior ingression of the plasma membrane.     

The peptidyl prolyl isomerase cyclophilin A localizes at the centrosome and the midbody and is required for cytokinesis

Failed cytokinesis leads to tetraploidy, which is an important intermediate preceding aneuploidy and the onset of tumorigenesis. The centrosome is required for the completion of cytokinesis through the transport of important components to the midbody; however, the identity of molecular components and the mechanism involved remains poorly understood. In this study, we report that the peptidyl prolyl isomerase cyclophilin A (cypA) is a centrosome protein that undergoes cell cycle-dependent relocation to the midzone and midbody during cytokinesis in Jurkat cells implicating a role during division. Depletion of cypA does not disrupt mitotic spindle formation or progression through anaphase; however, it leads to cytokinesis defects through an inability to resolve intercellular bridges, culminating in delayed or failed cytokinesis. Defective cytokinesis is also evident by an increased prevalence of midbody-arrested cells. Expression of wild-type cypA reverses the cytokinesis defect in knockout cells, whereas an isomerase mutant does not, indicating that the isomerisation activity of cypA is required for cytokinesis. In contrast, wild-type cypA and the isomerase mutant localize to the centrosome and midbody, suggesting that localization to these structures is independent of isomerase activity. Depletion of cypA also generates tetraploid cells and supernumerary centrosomes. Finally, colony formation in soft agar is impaired in cypA-knockout cells, suggesting that cypA confers clonogenic advantage on tumor cells. Collectively, this data reveals a novel role for cypA isomerase activity in the completion of cytokinesis and the maintenance of genome stability.     

Role of the midbody matrix in cytokinesis: RNAi and genetic rescue analysis of the mammalian motor protein CHO1

CHO1 is a kinesin-like motor protein essential for cytokinesis in mammalian cells. To analyze how CHO1 functions, we established RNAi and genetic rescue assays. CHO1-depleted cells reached a late stage of cytokinesis but fused back to form binucleate cells because of the absence of the midbody matrix in the middle of the intercellular bridge. Expression of exogenous CHO1 restored the formation of the midbody matrix and rescued cytokinesis in siRNA-treated cells. By analyzing phenotypes rescued with different constructs, it was shown that both motor and stalk domains function in midbody formation, whereas the tail is essential for completion of cytokinesis after the midbody matrix has formed. During the terminal stage of cytokinesis, different subregions of the tail play distinctive roles in stabilizing the midbody matrix and maintaining an association between the midbody and cell cortex. These results demonstrate that CHO1 consists of functionally differentiated subregions that act in concert to ensure complete cell separation.     

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.     

Vimentin dynamics during the mitogenic stimulation of mouse splenic lymphocytes

We have used double immunofluorescence and electron microscopy to examine the distribution of tubulin and vimentin during the stimulation of mouse splenic lymphocytes by the mitogen concanavalin A. In unstimulated cells, vimentin forms a filamentous network partially coincident with the radial pattern of microtubules. In stimulated cells, the numbers of microtubules assembled from the centrosome have increased and vimentin is organized as an aggregate located near the centrosome. When these cells enter mitosis, vimentin is arranged into a filamentous cage enclosing the mitotic apparatus. During cytokinesis, the polar centrosomes are observed at a position adjacent to the midbody and vimentin is detected as an aggregate, similar to that seen prior to mitosis, close to the centrosome in each daughter cell. Using several agents, such as colchicine, colcemid, nocodazole, and taxol, which affect microtubule assembly, we have observed that the vimentin system, although closely related spatially to the microtubule complex in lymphocytes, can still reorganize independently as these cells progress through the cell cycle. Throughout mitogenic stimulation in the continued presence of taxol, microtubules are reorganized into a few thick bundles while the vimentin system undergoes a sequence of rearrangements similar to those observed during normal stimulation. These data suggest that vimentin dynamics may be important in the progression of lymphocytes through the cell cycle in response to mitogen.     

Cell migration regulates the kinetics of cytokinesis

Cytokinesis is the final stage of cell division in which the daughter cells separate. Although a growing body of evidence suggests that cell migration-induced traction forces may be required to provide physical assistance for daughter cells to dissociate during abscission, the role of cell migration in cytokinesis has not been directly elucidated. Recently, we have demonstrated that Crk and paxillin, which are pivotal components of the cell migration machinery, localize to the midbody and are essential for the abscission. These findings provided an important link between the cell migration and cytokinesis machineries and prompted us to dissect the role of cell migration in cytokinesis. We show that cell migration controls the kinetics of cleavage furrowing, midbody extension and abscission and coordinates proper subcellular redistribution of Crk and syntaxin-2 to the midbody after ingression.Key words: cell migration, cytokinesis, midbody, abscission, cleavage furrow, Crk, paxillin, syntaxin-2, ExoT     

TAXOL REDUCES THE RATE OF CYTOKINESIS IN PtK1CELLS

Mitotic PtK₁cells were treated both during mid-anaphase and at furrow initiation with the potent microtubule (MT) stabilizing agent, taxol, to determine the role of MTs in the rate of cytokinetic events. Rates of cytokinesis (μm/min) were measured by changes in furrow diameter. Incubation of PtK₁cells during mid-anaphase with 5 μg/ml taxol slows the rate of cytokinesis by an average of 43%. Instead of furrow initiation to midbody formation taking an average of 10.7 min (1.6 μm/min), furrowing to midbody formation was completed in an average of 19.0 min (0.9 μm/min), which does not include the 7-min period between taxol application in mid-anaphase and furrow initiation. Application of 5 μg/ml taxol to cells at furrow initiation had a reduced effect on decreasing the rate of cytokinesis and midbody formation; furrowing to midbody formation took an average of 14.6 min (1.2 μm/min). These data suggest that delays in the rate of cytokinesis is dependent on the mitotic stage at which taxol is applied. Ultrastructural analysis shows that taxol treatment of anaphase cells prevents midbody formation during early G₁, yet MT number and organization in the furrowed region is not significantly altered from untreated cells. There is little change in the organization and amount of contractile ring microfilaments, yet filaments are also found parallel to midbody MTs. Our results may be explained by the fact that taxol tends to stabilize MTs which probably affects the rate at which they depolymerize in the terminal phases of cytokinesis. Reduction in depolymerization rates of a stable population of MTs could serve to regulate the rate of cytokinesis.     

Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Our previous studies show that microtubule motor CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signaling cascade. However, the molecular function of CENP-E at the midbody had remained elusive. Here we show that CENP-E interacts with Skp1 at the midbody and participates in cytokinesis. CENP-E interacts with Skp1 in vitro and in vivo via its coiled-coil domain. Our yeast two-hybrid assays mapped the binding interfaces to the central stalk region of CENP-E (955-1571 aa) and the C-terminal 33 amino acids of Skp1, respectively. Our immunocytochemical studies revealed that CENP-E targets to the midbody prior to Skp1 and the midbody localization of CENP-E becomes diminished as Skp1 arrives at the midbody. Suppression of Skp1 in mitotic HeLa cells by siRNA resulted in accumulation of telophase cells with elongated inter-cell bridges and with midbodies stretched 2-3 times longer than that of normal cells. These Skp1-eliminated or -suppressed cells accumulate higher level of CENP-E, suggesting that spatiotemporal regulation of CENP-E degradation at the midbody is essential for cytokinesis. Over-expression of Skp1 lacking the CENP-E-binding domain confirmed that Skp1-CENP-E interaction is essential for faithful cytokinesis. We hypothesize that CENP-E degradation is essential for faithful mitotic exit and the proteolysis of CENP-E is mediated by SCF via a direct Skp1 link.     

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.     

Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission

The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.     

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.     

A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin

O-linked β-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.     

The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring–derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.     

Vesicle trafficking and membrane remodelling in cytokinesis

All cells complete cell division by the process of cytokinesis. At the end of mitosis, eukaryotic cells accurately mark the site of division between the replicated genetic material and assemble a contractile ring comprised of myosin II, actin filaments and other proteins, which is attached to the plasma membrane. The myosin-actin interaction drives constriction of the contractile ring, forming a cleavage furrow (the so-called 'purse-string' model of cytokinesis). After furrowing is completed, the cells remain attached by a thin cytoplasmic bridge, filled with two anti-parallel arrays of microtubules with their plus-ends interdigitating in the midbody region. The cell then assembles the abscission machinery required for cleavage of the intercellular bridge, and so forms two genetically identical daughter cells. We now know much of the molecular detail of cytokinesis, including a list of potential genes/proteins involved, analysis of the function of some of these proteins, and the temporal order of their arrival at the cleavage site. Such studies reveal that membrane trafficking and/or remodelling appears to play crucial roles in both furrowing and abscission. In the present review, we assess studies of vesicular trafficking during cytokinesis, discuss the role of the lipid components of the plasma membrane and endosomes and their role in cytokinesis, and describe some novel molecules implicated in cytokinesis. The present review covers experiments performed mainly on tissue culture cells. We will end by considering how this mechanistic insight may be related to cytokinesis in other systems, and how other forms of cytokinesis may utilize similar aspects of the same machinery.     

Katanin p60 Contributes to Microtubule Instability around the Midbody and Facilitates Cytokinesis in Rat Cells

The completion of cytokinesis is crucial for mitotic cell division. Cleavage furrow ingression is followed by the breaking and resealing of the intercellular bridge, but the detailed mechanism underlying this phenomenon remains unknown. Katanin is a microtubule-severing protein comprised of an AAA ATPase subunit and an accessory subunit designated as p60 and p80, respectively. Localization of katanin p60 was observed at the midzone to midbody from anaphase to cytokinesis in rat cells, and showed a ring-shaped distribution in the gap between the inside of the contractile ring and the central spindle bundle in telophase. Katanin p60 did not bind with p80 at the midzone or midbody, and localization was shown to be dependent on microtubules. At the central spindle and the midbody, no microtubule growth plus termini were seen with katanin p60, and microtubule density was inversely correlated with katanin p60 density in the region of katanin p60 localization that seemed to lead to microtubule destabilization at the midbody. Inhibition of katanin p60 resulted in incomplete cytokinesis by regression and thus caused the appearance of binucleate cells. These results suggest that katanin p60 contributes to microtubule instability at the midzone and midbody and facilitates cytokinesis in rat cells.