Multiple centrosomes in tumor cells create the potential for multipolar divisions that can lead to aneuploidy and cell death. Nevertheless, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses. A genome-wide RNAi screen in Drosophila S2 cells and a secondary analysis in cancer cells defined mechanisms that suppress multipolar mitoses. In addition to proteins that organize microtubules at the spindle poles, we identified novel roles for the spindle assembly checkpoint, cortical actin cytoskeleton, and cell adhesion. Using live cell imaging and fibronectin micropatterns, we found that interphase cell shape and adhesion pattern can determine the success of the subsequent mitosis in cells with extra centrosomes. These findings may identify cancer-selective therapeutic targets: HSET, a normally nonessential kinesin motor, was essential for the viability of certain extra centrosome-containing cancer cells. Thus, morphological features of cancer cells can be linked to unique genetic requirements for survival.[Keywords: Centrosomes; mitosis; actin; adhesion; cancer; cell cycle] Supplemental material is available at http://www.genesdev.org.

It is well established that multiple microtubule-based motors contribute to the formation and function of the mitotic spindle, but how the activities of these motors interrelate remains unclear.Here we visualize spindle formation in living Drosophila embryos to show that spindle pole movements are directed by a temporally coordinated balance of forces generated by three mitotic motors, cytoplasmic dynein, KLP61F, and Ncd. Specifically, our findings suggest that dynein acts to move the poles apart throughout mitosis and that this activity is augmented by KLP61F after the fenestration of the nuclear envelope, a process analogous to nuclear envelope breakdown, which occurs at the onset of prometaphase. Conversely, we find that Ncd generates forces that pull the poles together between interphase and metaphase, antagonizing the activity of both dynein and KLP61F and serving as a brake for spindle assembly. During anaphase, however, Ncd appears to have no effect on spindle pole movements, suggesting that its activity is downregulated at this time, allowing dynein and KLP61F to drive spindle elongation during anaphase B. INTRODUCTIONThe segregation of chromosomes during mitosis depends on the action of a self-organizing, bipolar machine called the mitotic spindle. It is now established that the formation and function of the mitotic spindle requires numerous microtubule (MT)-based motor proteins (Hoyt and Geiser, 1996;Vale and Fletterick, 1997). Although the identities of many of these mitotic motors are becoming clear, their specific functional interrelationships have been extremely difficult to ascertain.Among all mitotic movements, the positioning of spindle poles during the assembly and elongation of the bipolar mitotic spindle may require the greatest degree of cooperation between different motors. This process is particularly complex because it occurs in a pathway consisting of several, temporally distinct stages, during which the organization of spindle microtubules and the general environment of the cell change dramatically (McIntosh and McDonald, 1989). The members of at least three families of MT motors are thought to play important roles in this pathway. These are the bipolar kinesins, the C-terminal kinesins, and cytoplasmic dynein.The bipolar (or BimC) kinesins (Vale and Fletterick, 1997) comprise a family of plus-end-directed motors, which have a bipolar morphology with motor domains at both ends of a central rod (Cole et al., 1994; Kashina et al., 1996a,b; Gordon and Roof, 2000). Functionally, these motors are thought to play a role in either the assembly or maintenance of spindle bipolarity, because their inhibition results in the formation of monopolar mitotic spindles (Enos and Morris, 1990;Hagan and Yanagida, 1990;Roof et al., 1991;Hoyt et al., 1992;Sawin et al., 1992;Heck et al., 1993;Blangy et al., 1995;Sharp et al., 1999b). Support for a role for bipolar kinesins in spindle maintenance but not assembly comes from the recent findings that inhibiting the Drosophila bipolar kinesin KLP61F does not pr...

It has been proposed that the suppression of poleward flux within interpolar microtubule (ipMT) bundles of Drosophila embryonic spindles couples outward forces generated by a sliding filament mechanism to anaphase spindle elongation. Here, we (i) propose a molecular mechanism in which the bipolar kinesin KLP61F persistently slides dynamically unstable ipMTs outward, the MT depolymerase KLP10A acts at the poles to convert ipMT sliding to flux, and the chromokinesin KLP3A inhibits the depolymerase to suppress flux, thereby coupling ipMT sliding to spindle elongation; (ii) used KLP3A inhibitors to interfere with the coupling process, which revealed an inverse linear relation between the rates of flux and elongation, supporting the proposed mechanism and demonstrating that the suppression of flux controls both the rate and onset of spindle elongation; and (iii) developed a mathematical model using force balance and rate equations to describe how motors sliding the highly dynamic ipMTs apart can drive spindle elongation at a steady rate determined by the extent of suppression of flux.C hromosome segregation depends upon the action of the spindle, a protein machine that uses ensembles of kinesin and dynein motors plus microtubule (MT) dynamics to move chromatids polewards (anaphase A) and to elongate the spindle (anaphase B) (1). Anaphase B is driven in part by a bipolar kinesin-dependent sliding filament mechanism (2-9), with the extent of spindle elongation determined by MT polymerization in the overlap zone (2). Poleward flux, the movement of tubulin subunits from the MT plus ends facing the spindle equator to their minus ends at the poles (10-14), is proposed to constrain the length of metaphase spindles, with subsequent inhibition of depolymerization at the poles converting metaphase flux to anaphase spindle elongation (12,15,16).In support of this hypothesis, we observed that a suppression of poleward flux occurs at anaphase B onset: tubulin speckles within interpolar MTs (ipMTs) of Drosophila embryonic spindles fluxed toward the stationary poles of preanaphase B (herein meaning metaphase-anaphase A) spindles, but during anaphase B the speckles moved apart at the same rate as the poles (12). Here, we propose that three mitotic motors play critical roles in this process, based on previous studies (Fig. 1A). First, the bipolar kinesin KLP61F drives a sliding filament mechanism that underlies spindle elongation, because inhibiting KLP61F (in an Ncd-null mutant to circumvent the collapse of prometaphase spindles) inhibits anaphase B (9). Second, the kin I kinesin KLP10A depolymerizes ipMTs at the poles of preanaphase spindles, converting sliding to poleward flux; its inhibition leads to the premature suppression of flux and spindle elongation (14), suggesting that it is down-regulated at the onset of anaphase B. Finally, the chromokinesin KLP3A organizes ipMTs into bundles and is required for efficient anaphase spindle elongation (17).Here, we report experimental and theoretical results that provide a quantitative d...

Defects in the architecture or integrity of the nuclear envelope are associated with a variety of human diseases. Micronuclei, one common nuclear aberration, are an origin for chromothripsis, a catastrophic mutational process that is commonly observed in cancer. Chromothripsis occurs after micronuclei spontaneously lose nuclear envelope integrity, which generates chromosome fragmentation. Disruption of the nuclear envelope exposes DNA to the cytoplasm and initiates innate immune proinflammatory signalling. Despite its importance, the basis of the fragility of the micronucleus nuclear envelope  is not known. Here we show that micronuclei undergo defective nuclear envelope assembly. Only 'core' nuclear envelope proteins assemble efficiently on lagging chromosomes, whereas 'non-core' nuclear envelope proteins, including nuclear pore complexes (NPCs), do not. Consequently, micronuclei fail to properly import key proteins that are necessary for the integrity of the nuclear envelope and genome. We show that spindle microtubules block assembly of NPCs and other non-core nuclear envelope proteins on lagging chromosomes, causing an irreversible defect in nuclear envelope assembly. Accordingly, experimental manipulations that position missegregated chromosomes away from the spindle correct defective nuclear envelope assembly, prevent spontaneous nuclear envelope disruption, and suppress DNA damage in micronuclei. Thus, during mitotic exit in metazoan cells, chromosome segregation and nuclear envelope assembly are only loosely coordinated by the timing of mitotic spindle disassembly. The absence of precise checkpoint controls may explain why errors during mitotic exit are frequent and often trigger catastrophic genome rearrangements.

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, it is expected the development of a potent, selective small molecule inhibitor of Mps1 would greatly facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 co-crystal structures of novel, selective small molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit, and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells possessing extra centrosomes, an abnormality found in some cancers, Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

Precise control of centrosome number is crucial for bipolar spindle assembly and accurate transmission of genetic material to daughter cells. Failure to properly control centrosome number results in supernumerary centrosomes, which are frequently found in cancer cells. This presents a paradox: during mitosis, cells with more than two centrosomes are prone to multipolar mitoses and cell death, however, cancer cells possessing extra centrosomes usually divide successfully. One mechanism frequently utilized by cancer cells to escape death caused by multipolar mitoses is the clustering of supernumerary centrosomes into bipolar arrays. An understanding of the molecular mechanisms by which cancer cells can suppress multipolar mitoses is beginning to emerge. Here, we review what's currently known about centrosome clustering mechanisms and discuss potential strategies to target these mechanisms for the selective killing of cancer cells.

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The defining characteristic of a tumor cell is its ability to escape the constraints imposed by neighboring cells, invade the surrounding tissue and metastasize to distant sites. This invasive property of tumor cells is dependent on activation of proteinases at the cell surface. The serine proteinase plasmin is one of the key proteinases that participate in the pericellular proteolysis associated with the invasive program of tumor cells. The assembly of plasminogen and tissue plasminogen activator at the endothelial cell surface or on the fibrin clot provides a focal point for plasmin generation and therefore plays an important role in maintaining blood fluidity and promoting fibrinolysis. S100A10, a member of the S100 family of Ca2+-binding proteins, is a dimeric protein composed of two 11 kDa subunits. Typically, S100A10 is found in most cells bound to its annexin A2 ligand as the heterotetrameric (S100A10)2(annexin A2)2 complex, AIIt. In addition to an intracellular distribution, S100A10 is present on the extracellular surface of many cells. The carboxyl-terminal lysines of S100A10 bind tPA and plasminogen resulting in the stimulation of tPA-dependent plasmin production. Carboxypeptidases cleave the carboxyl-terminal lysines of S100A10, resulting in a loss of binding and activity. Plasmin binds to S100A10 at a distinct site and the formation of the S100A10-plasmin complex stimulates plasmin autoproteolysis thereby providing a highly localized transient pulse of plasmin activity at the cell surface. The binding of tPA and plasmin to S100A10 also protects against inhibition by physiological inhibitors, PAI-1 and alpha2-antiplasmin, respectively. S100A10 also colocalizes plasminogen with the uPA-uPAR complex thereby localizing and stimulating uPA-dependent plasmin formation to the surface of cancer cells. The loss of S100A10 from the extracellular surface of cancer cells results in a significant loss in plasmin generation. In addition, S100A10 knock-down cells demonstrate a dramatic loss in extracellular matrix degradation and invasiveness as well as reduced metastasis. Annexin A2 plays an important role in plasminogen regulation by controlling the levels of extracellular S100A10 and by acting as a plasmin reductase. The mechanism by which annexin A2 regulates the extracellular levels of S100A10 is unknown. This review highlights the important part that S100A10 plays in plasmin regulation and the role this protein plays in cancer cell invasiveness and metastasis.