C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times during oocyte meiotic spindle assembly and promote both spindle pole coalescence and stability.

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.

Aurora kinase A is essential for meiosis in mouse oocytes.

The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile, and their oocytes fail to complete meiosis I. In determining AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at acentriolar microtubule organizing centers (aMTOCs; meiotic spindle poles). This activation induces fragmentation of the aMTOCs, a step essential for building a bipolar spindle. We also show that AURKA is required for regulating localization of TACC3, another protein required for spindle building. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.

Loss of acentriolar MTOCs disrupts spindle pole Aurora A and assembly of the liquid-like meiotic spindle domain in oocytes.

Oocyte-specific knockdown of pericentrin (PCNT) in transgenic (Tg) mice disrupts acentriolar microtubule-organizing center (aMTOC) formation, leading to spindle instability and error-prone meiotic division. Here, we show that PCNT-depleted oocytes lack phosphorylated Aurora A (pAURKA) at spindle poles, while overall levels are unaltered. To test aMTOC-associated AURKA function, metaphase II (MII) control (WT) and Tg oocytes were briefly exposed to a specific AURKA inhibitor (MLN8237). Similar defects were observed in Tg and MLN8237-treated WT oocytes, including altered spindle structure, increased chromosome misalignment and impaired microtubule regrowth. Yet, AURKA inhibition had a limited effect on Tg oocytes, revealing a critical role for aMTOC-associated AURKA in regulating spindle stability. Notably, spindle instability was associated with disrupted γ-tubulin and lack of the liquid-like meiotic spindle domain (LISD) in Tg oocytes. Analysis of this Tg model provides the first evidence that LISD assembly depends expressly on aMTOC-associated AURKA, and that Ran-mediated spindle formation ensues without the LISD. These data support that loss of aMTOC-associated AURKA and failure of LISD assembly contribute to error-prone meiotic division in PCNT-depleted oocytes, underscoring the essential role of aMTOCs for spindle stability.

Mms19 promotes spindle microtubule assembly in Drosophila neural stem cells.

Mitotic divisions depend on the timely assembly and proper orientation of the mitotic spindle. Malfunctioning of these processes can considerably delay mitosis, thereby compromising tissue growth and homeostasis, and leading to chromosomal instability. Loss of functional Mms19 drastically affects the growth and development of mitotic tissues in Drosophila larvae and we now demonstrate that Mms19 is an important factor that promotes spindle and astral microtubule (MT) growth, and MT stability and bundling. Mms19 function is needed for the coordination of mitotic events and for the rapid progression through mitosis that is characteristic of neural stem cells. Surprisingly, Mms19 performs its mitotic activities through two different pathways. By stimulating the mitotic kinase cascade, it triggers the localization of the MT regulatory complex TACC/Msps (Transforming Acidic Coiled Coil/Minispindles, the homolog of human ch-TOG) to the centrosome. This activity of Mms19 can be rescued by stimulating the mitotic kinase cascade. However, other aspects of the Mms19 phenotypes cannot be rescued in this way, pointing to an additional mechanism of Mms19 action. We provide evidence that Mms19 binds directly to MTs and that this stimulates MT stability and bundling.

Spindle pole power in health and disease.

Saccharomyces cerevisiae has been widely used as a model system for the study of basic biological processes which are usually evolutionarily conserved from yeasts to multicellular eukaryotes. These studies are very important because they shed light on mechanisms that are altered in human diseases and help the development of new biomarkers and therapies. The mitotic spindle is a conserved apparatus that governs chromosome segregation during mitosis. Given its crucial role for genome stability and, therefore, for cell viability, its structure and function are strictly regulated. Recent findings reveal new levels of regulation in mitotic spindle dynamics and link spindle pole diversification with cell fate determination, health, disease and aging.

Stathmin/Op18 depletion induces genomic instability and leads to premature senescence in human normal fibroblasts.

Stathmin/oncoprotein18 regulates microtubule dynamics and participates in mitotic entry and exit. We isolated stathmin as a physically interacting partner of KIFC1, a minus-end-directed kinesin functioning in bipolar spindle formation and maintenance. We found that stathmin depletion leads to multipolar spindle formation in IMR-90 normal human fibroblasts. Stathmin-depleted IMR-90 cells showed early mitotic delay but managed to undergo chromosome segregation by forming multiple poles or pseudo-bipoles. Consistent with these observations, lagging chromosomes, and micronuclei were elevated in stathmin-depleted IMR-90 cells, demonstrating that stathmin is essential for maintaining genomic stability during mitosis in human cells. Genomic instability induced by stathmin depletion led to premature senescence without any indication of cell death in normal IMR-90 cells. Double knock-down of both stathmin and p53 also did not induce cell death in IMR-90 cells, while the stathmin knock-down triggered apoptosis in p53-proficient human lung adenocarcinoma cells. Our results suggest that stathmin is essential in bipolar spindle formation to maintain genomic stability during mitosis, and the depletion of stathmin prevents the initiation of chromosome instability by inducing senescence in human normal fibroblasts.

Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

Asymmetry of the budding yeast Tem1 GTPase at spindle poles is required for spindle positioning but not for mitotic exit.

The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.

TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis.

Dynamic changes in spindle structure and function are essential for maintaining genomic integrity during the cell cycle. Spindle dynamics are highly dependent on several microtubule-associated proteins that coordinate the dynamic behavior of microtubules, including microtubule assembly, stability and organization. Here, we show that translationally controlled tumor protein (TCTP) is a novel microtubule-associated protein that regulates spindle dynamics during meiotic maturation. TCTP was expressed and widely distributed in the cytoplasm with strong enrichment at the spindle microtubules during meiosis. TCTP was found to be phosphorylated during meiotic maturation, and was exclusively localized to the spindle poles. Knockdown of TCTP impaired spindle organization without affecting chromosome alignment. These spindle defects were mostly due to the destabilization of the polar microtubules. However, the stability of kinetochore microtubules attached to chromosomes was not affected by TCTP knockdown. Overexpression of a nonphosphorylable mutant of TCTP disturbed meiotic maturation, stabilizing the spindle microtubules. In addition, Plk1 was decreased by TCTP knockdown. Taken together, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle microtubule dynamics during meiotic maturation in mouse oocytes.

Asymmetric spindle pole formation in CPAP-depleted mitotic cells.

CPAP is an essential component for centriole formation. Here, we report that CPAP is also critical for symmetric spindle pole formation during mitosis. We observed that pericentriolar material between the mitotic spindle poles were asymmetrically distributed in CPAP-depleted cells even with intact numbers of centrioles. The length of procentrioles was slightly reduced by CPAP depletion, but the length of mother centrioles was not affected. Surprisingly, the young mother centrioles of the CPAP-depleted cells are not fully matured, as evidenced by the absence of distal and subdistal appendage proteins. We propose that the selective absence of centriolar appendages at the young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells. Our results suggest that the neural stem cells with CPAP mutations might form asymmetric spindle poles, which results in premature initiation of differentiation.