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...

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.

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...