Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-␣-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.

Aberrant cell division is a hallmark of cancer, but the molecular circuitries of this process in tumor cells are not well understood. Here, we used a high-throughput proteomics screening to identify novel molecular partners of survivin, an essential regulator of mitosis overexpressed in cancer. We found that survivin associates with the small GTPase Ran in an evolutionarily conserved recognition in mammalian cells and Xenopus laevis extracts. This interaction is regulated during the cell cycle, involves Ran-GTP, requires a discrete binding interface centered on Glu65 in survivin, and is independent of the Ran effector Crm1. Disruption of a survivin-Ran complex does not affect the assembly of survivin within the chromosomal passenger complex or its cytosolic accumulation, but it inhibits the delivery of the Ran effector molecule TPX2 to microtubules. In turn, this results in aberrant mitotic spindle formation and chromosome missegregation in tumor, but not normal, cells. Therefore, survivin is a novel effector of Ran signaling, and this pathway may be preferentially exploited for spindle assembly in tumor cells.Deregulated cell division is a hallmark of cancer (31), which results in unrestrained cell proliferation, abrogation of cell cycle checkpoints, and propensity to aneuploidy (27). These processes hinge on spatial-temporal assembly of a bipolar mitotic spindle (23), where microtubules nucleating from duplicated centrosomes or assembled in proximity of mitotic chromosomes "search and capture" chromatids and ultimately segregate them between daughter cells (46). Reconstitution experiments in model organisms, particularly cycled Xenopus laevis extracts, uncovered a pivotal role of the small GTPase Ran in the mechanism of spindle formation (36). We now know that a gradient of Ran-GTP assembles on mitotic chromosomes (20), largely through the chromatin-associated activity of the nucleotide exchange factor RCC1. In turn, this releases Ran effector molecules implicated in spindle formation (9, 15), including microtubule-stabilizing TPX2 (14), from an inhibitory interaction with importin ␣/␤ receptors (16, 45). Depletion of Ran in Xenopus extracts (9) or mammalian cells (15) or targeting its effector molecules (26, 41, 43) profoundly impairs spindle formation, causing the appearance of flattened mitotic spindles, severely depleted of microtubules, and abnormal chromosomal segregation.Among the regulators of cell division aberrantly overexpressed in cancer is survivin (3), a member of the inhibitor of apoptosis (IAP) gene family with dual roles in suppression of cell death and control of mitosis (1, 29). The latter has been linked to a multiplicity of functions, including kinetochore targeting of the chromosomal passenger complex (22), enhancement of Aurora B kinase activity (7), control of kinetochore-microtubule interactions for proper chromosomal alignment (39), participation in the spindle assembly checkpoint (11), and regulation of microtubule dynamics for spindle formation (37). Although these functions are ess...

Survivin is a member of the inhibitor of apoptosis (IAP) protein family that serves critical roles in mitosis and cytokinesis. Many studies have suggested Survivin's involvement in spindle regulation, but direct biochemical evidence for this has been lacking. Using the cell-free system of Xenopus egg extracts, we tested whether Survivin was necessary for the assembly of metaphase spindles. Removal or inhibition of Xenopus Survivin causes the disruption in the formation of metaphase spindles. In particular, we observe the generation of microtubule (MT) asters or poorly formed shortened spindle structures. In the latter phenotype the spindle structures display a decrease pole-to-pole length and a reduction of MTs around the chromatin indicating that Survivin may promote the stabilization of MT-chromatin interactions. In addition, function analysis of Survivin's conserved phosphorylation site Thr34 (Thr43 in Xenopus) and tubulin-binding domain was also assessed in regulating spindle assembly. Treatment of Xenopus egg extracts with a recombinant Survivin mutant that contained an alanine residue substitution at Thr43 (SURT43A mutant) or that was missing the C-terminal tubulin-binding domain (SURCL mutant) produced an increased frequency of MT asters and shorten abnormal spindle structures in Xenopus egg extracts. Interestingly, a phosphomimetic mutation made at residue Thr43 of Survivin (SURT43E mutant) generated a high frequency of MT asters implying that premature 'activation' of Survivin may interfere with an early stage of spindle assembly. Taken together, we propose that Survivin is a necessary component of the mitotic spindle and its phosphorylation at residue Thr43 is important for Survivin function in spindle assembly.

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