Male meiotic spindle poles are stabilized by TACC3 and cKAP5/chTOG differently from female meiotic or somatic mitotic spindles in mice.

Transforming acidic acid coiled-coil protein 3 (TACC3) and cytoskeleton associated protein 5 (cKAP5; or colonic hepatic tumor overexpressed gene, chTOG) are vital for spindle assembly and stabilization initiated through TACC3 Aurora-A kinase interaction. Here, TACC3 and cKAP5/chTOG localization with monospecific antibodies is investigated in eGFP-centrin-2- expressing mouse meiotic spermatocytes. Both proteins bind spermatocyte spindle poles but neither kinetochore nor interpolar microtubules, unlike in mitotic mouse fibroblasts or female meiotic oocyte spindles. Spermatocytes do not display a liquid-like spindle domain (LISD), although fusing them into maturing oocytes generates LISD-like TACC3 condensates around sperm chromatin but sparse microtubule assembly. Microtubule inhibitors do not reduce TACC3 and cKAP5/chTOG spindle pole binding. MLN 8237 Aurora-A kinase inhibitor removes TACC3, not cKAP5/chTOG, disrupting spindle organization, chromosome alignment, and impacting spindle pole γ-tubulin intensity. The LISD disruptor 1,6-hexanediol abolished TACC3 in spermatocytes, impacting spindle bipolarity and chromosome organization. Cold microtubule disassembly and rescue experiments in the presence of 1,6-hexanediol reinforce the concept that spermatocyte TACC3 spindle pole presence is not required for spindle pole microtubule assembly. Collectively, meiotic spermatocytes without a LISD localize TACC3 and cKAP5/chTOG exclusively at spindle poles to support meiotic spindle pole stabilization during male meiosis, different from either female meiosis or mitosis.

RNF20 Regulates Oocyte Meiotic Spindle Assembly by Recruiting TPM3 to Centromeres and Spindle Poles.

Previously a ring finger protein 20 (RNF20) is found to be essential for meiotic recombination and mediates H2B ubiquitination during spermatogenesis. However, its role in meiotic division is still unknown. Here, it is shown that RNF20 is localized at both centromeres and spindle poles, and it is required for oocyte acentrosomal spindle organization and female fertility. RNF20-depleted oocytes exhibit severely abnormal spindle and chromosome misalignment caused by defective bipolar organization. Notably, it is found that the function of RNF20 in spindle assembly is not dependent on its E3 ligase activity. Instead, RNF20 regulates spindle assembly by recruiting tropomyosin3 (TPM3) to both centromeres and spindle poles with its coiled-coil motif. The RNF20-TPM3 interaction is essential for acentrosomal meiotic spindle assembly. Together, the studies uncover a novel function for RNF20 in mediating TPM3 recruitment to both centromeres and spindle poles during oocyte spindle assembly.

Endosulfine alpha maintains spindle pole integrity by recruiting Aurora A during mitosis.

BACKGROUND: The maintenance of spindle pole integrity is essential for spindle assembly and chromosome segregation during mitosis. However, the underlying mechanisms governing spindle pole integrity remain unclear. METHODS: ENSA was inhibited by siRNA or MKI-2 treatment and its effect on cell cycle progression, chromosome alignment and microtubule alignment was observed by immunohistochemical staining and western blotting. PP2A-B55α knockdown by siRNA was performed to rescue the phenotype caused by ENSA inhibition. The interaction between ENSA and Aurora A was detected by in situ PLA. Furthermore, orthotopic implantation of 4Tl-luc cancer cells was conducted to confirm the consistency between the in vitro and in vivo relationship of the ENSA-Aurora A interaction. RESULTS: During mitosis, p-ENSA is localized at the spindle poles, and the inhibition of ENSA results in mitotic defects, such as misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome defects, with a delay in mitotic progression. Although the mitotic delay caused by ENSA inhibition was rescued by PP2A-B55α depletion, spindle pole defects persisted. Notably, we observed a interaction between ENSA and Aurora A during mitosis, and inhibition of ENSA reduced Aurora A expression at the mitotic spindle poles. Injecting MKI-2-sensitized tumors led to increased chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. CONCLUSIONS: These findings provide novel insights into the regulation of spindle pole integrity by the MASTL-ENSA-Aurora A pathway during mitosis, highlighting the significance of ENSA in recruiting Aurora A to the spindle pole, independent of PP2A-B55α.

Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.

The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.

MRCK activates mouse oocyte myosin II for spindle rotation and male pronucleus centration.

Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning triggers cortical polarization, including the build-up of an actin cap surrounded by a ring of activated myosin II. While the role of the actin cap in promoting polar body formation is established, ring myosin II activation mechanisms and functions have remained elusive. Here, we show that ring myosin II activation requires myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), downstream of polarized Cdc42. MRCK inhibition resulted in spindle rotation defects during anaphase II, precluding polar body extrusion. Remarkably, disengagement of segregated chromatids from the anaphase spindle could rescue rotation. We further show that the MRCK/myosin II pathway is activated in the fertilization cone and is required for male pronucleus migration toward the center of the zygote. These findings provide novel insights into the mechanism of myosin II activation in oocytes and its role in orchestrating asymmetric division and pronucleus centration.

Recruitment of the lipid kinase Mss4 to the meiotic spindle pole promotes prospore membrane formation in Saccharomyces cerevisiae.

Spore formation in the budding yeast, Saccharomyces cerevisiae, involves de novo creation of four prospore membranes, each of which surrounds a haploid nucleus resulting from meiosis. The meiotic outer plaque (MOP) is a meiosis-specific protein complex associated with each meiosis II spindle pole body (SPB). Vesicle fusion on the MOP surface creates an initial prospore membrane anchored to the SPB. Ady4 is a meiosis-specific MOP component that stabilizes the MOP-prospore membrane interaction. We show that Ady4 recruits the lipid kinase, Mss4, to the MOP. MSS4 overexpression suppresses the ady4∆ spore formation defect, suggesting that a specific lipid environment provided by Mss4 promotes maintenance of prospore membrane attachment to MOPs. The meiosis-specific Spo21 protein is an essential structural MOP component. We show that the Spo21 N terminus contains an amphipathic helix that binds to prospore membranes. A mutant in SPO21 that removes positive charges from this helix shares phenotypic similarities to ady4∆. We propose that Mss4 generates negatively charged lipids in prospore membranes that enhance binding by the positively charged N terminus of Spo21, thereby providing a mechanism by which the MOP-prospore membrane interaction is stabilized.

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles.

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 To prevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

Aurora A phosphorylates Ndel1 to reduce the levels of Mad1 and NuMA at spindle poles.

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally absent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters in Xenopus extracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.

PCMD-1 bridges the centrioles and the pericentriolar material scaffold in C. elegans.

Correct cell division relies on the formation of a bipolar spindle. In animal cells, microtubule nucleation at the spindle poles is facilitated by the pericentriolar material (PCM), which assembles around a pair of centrioles. Although centrioles are essential for PCM assembly, the proteins that anchor the PCM to the centrioles are less known. Here, we investigate the molecular function of PCMD-1 in bridging the PCM and the centrioles in Caenorhabditis elegans. We demonstrate that the centrosomal recruitment of PCMD-1 is dependent on the outer centriolar protein SAS-7. The most C-terminal part of PCMD-1 is sufficient to target it to the centrosome, and the coiled-coil domain promotes its accumulation by facilitating self-interaction. We reveal that PCMD-1 interacts with the PCM scaffold protein SPD-5, the mitotic kinase PLK-1 and the centriolar protein SAS-4. Using an ectopic translocation assay, we show that PCMD-1 can selectively recruit downstream PCM scaffold components to an ectopic location in the cell, indicating that PCMD-1 is able to anchor the PCM scaffold proteins at the centrioles. Our work suggests that PCMD-1 is an essential functional bridge between the centrioles and the PCM.

Forcing dividing cancer cells to die; low-dose drug combinations to prevent spindle pole clustering.

Mitosis, under the control of the microtubule-based mitotic spindle, is an attractive target for anti-cancer treatments, as cancer cells undergo frequent and uncontrolled cell divisions. Microtubule targeting agents that disrupt mitosis or single molecule inhibitors of mitotic kinases or microtubule motors kill cancer cells with a high efficacy. These treatments have, nevertheless, severe disadvantages: they also target frequently dividing healthy tissues, such as the haematopoietic system, and they often lose their efficacy due to primary or acquired resistance mechanisms. An alternative target that has emerged in dividing cancer cells is their ability to "cluster" the poles of the mitotic spindle into a bipolar configuration. This mechanism is necessary for the specific survival of cancer cells that tend to form multipolar spindles due to the frequent presence of abnormal centrosome numbers or other spindle defects. Here we discuss the recent development of combinatorial treatments targeting spindle pole clustering that specifically target cancer cells bearing aberrant centrosome numbers and that have the potential to avoid resistance mechanism due their combinatorial nature.

The spindle pole-body localization of activated cytoplasmic dynein is cell cycle-dependent in Aspergillus nidulans.

Cytoplasmic dynein is a minus end-directed microtubule motor that can be activated by cargo adapters. In Aspergillus nidulans, overexpression of ΔC-HookA, the early endosomal adapter HookA missing its cargo-binding site, causes activated dynein to accumulate at septa and spindle pole bodies (SPBs) where the microtubule-organizing centers are located. Intriguingly, only some interphase nuclei show SPB signals of dynein. Here we present data demonstrating that localization of the activated dynein at SPBs is cell cycle-dependent: SPB dynein signals are seen to associate with nuclei at early G1 but disappear at about the G1-S boundary.

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly.

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes.

Spindle assembly is spatially regulated by a chromosome-derived Ran- GTP gradient. Previous work proposed that Ran-GTP activates spindle assembly factors (SAFs) around chromosomes by dissociating inhibitory importins from SAFs. However, it is unclear whether the Ran-GTP gradient equivalently activates SAFs that localize at distinct spindle regions. In addition, Ran's dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic depletion assays to dissect Ran's mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle-pole focusing or for targeting TPX2. On the other hand, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions to set proper spindle length during prometaphase. We demonstrated that Ran-GTP and importin-ß coordinately promote HURP's dynamic microtubule binding-dissociation cycle, which maintains HURP near chromosomes during metaphase. Together, we propose that the Ran pathway acts on spindle assembly independently of its interphase functions in mitotic human cells but does not equivalently regulate all Ran-regulated SAFs. Ran-dependent spindle assembly is likely coupled with additional parallel pathways that activate SAFs distantly located from the chromosomes.

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.

Methylglyoxal inhibits nuclear division through alterations in vacuolar morphology and accumulation of Atg18 on the vacuolar membrane in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and it inhibits the growth of cells in all kinds of organisms. We recently reported that MG inhibits nuclear division in Saccharomyces cerevisiae. However, the mechanism by which MG blocks nuclear division remains unclear. Here, we show that increase in the levels of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is crucial for the inhibitory effects of MG on nuclear division, and the deletion of PtdIns(3,5)P2-effector Atg18 alleviated the MG-mediated inhibitory effects. Previously, we reported that MG altered morphology of the vacuole to a single swelling form, where PtdIns(3,5)P2 accumulates. The changes in the vacuolar morphology were also needed by MG to exert its inhibitory effects on nuclear division. The known checkpoint machinery, including the spindle assembly checkpoint and morphological checkpoint, are not involved in the blockade of nuclear division by MG. Our results suggest that both the accumulation of Atg18 on the vacuolar membrane and alterations in vacuolar morphology are necessary for the MG-induced inhibition of nuclear division.

AKAP350 enables p150glued /EB1 interaction at the spindle poles.

A-kinase anchoring protein 350 (AKAP350) is a centrosomal/Golgi scaffold protein, critical for the regulation of microtubule dynamics. AKAP350 recruits end-binding protein 1 (EB1) to the centrosome in mitotic cells, ensuring proper spindle orientation in epithelial cells. AKAP350 also interacts with p150glued, the main component of the dynactin complex. In the present work, we found that AKAP350 localized p150glued to the spindle poles, facilitating p150glued/EB1 interaction at these structures. Our results further showed that the decrease in AKAP350 expression reduced p150glued localization at astral microtubules and impaired the elongation of astral microtubules during anaphase. Overall, this study provides mechanistic data on how microtubule regulatory proteins gather to define microtubule dynamics in mitotic cells.

Microtubule assembly and pole coalescence: early steps in Caenorhabditiselegans oocyte meiosis I spindle assembly.

How oocytes assemble bipolar meiotic spindles in the absence of centrosomes as microtubule organizing centers remains poorly understood. We have used live cell imaging in Caenorhabditis elegans to investigate requirements for the nuclear lamina and for conserved regulators of microtubule dynamics during oocyte meiosis I spindle assembly, assessing these requirements with respect to recently identified spindle assembly steps. We show that the nuclear lamina is required for microtubule bundles to form a peripheral cage-like structure that appears shortly after oocyte nuclear envelope breakdown and surrounds the oocyte chromosomes, although bipolar spindles still assembled in its absence. Although two conserved regulators of microtubule nucleation, RAN-1 and γ-tubulin, are not required for bipolar spindle assembly, both contribute to normal levels of spindle-associated microtubules and spindle assembly dynamics. Finally, the XMAP215 ortholog ZYG-9 and the nearly identical minus-end directed kinesins KLP-15/16 are required for proper assembly of the early cage-like structure of microtubule bundles, and for early spindle pole foci to coalesce into a bipolar structure. Our results provide a framework for assigning molecular mechanisms to recently described steps in C. elegans oocyte meiosis I spindle assembly.

Mis12 controls cyclin B1 stabilization via Cdc14B-mediated APC/CCdh1 regulation during meiotic G2/M transition in mouse oocytes.

Mammalian oocytes are arrested at G2/prophase of the first meiosis. After a hormone surge, oocytes resume meiosis, undergoing germinal vesicle breakdown (GVBD). This process is regulated by Cdk1/cyclin B1. Here, we report that Mis12 is required for G2/M transition by regulating cyclin B1 accumulation via Cdc14B-mediated APC/CCdh1 regulation, but is not essential for spindle and chromosome dynamics during meiotic maturation. Depletion of Mis12 severely compromised GVBD by impairing cyclin B1 accumulation. Importantly, impaired GVBD after Mis12 depletion was rescued not only by overexpressing cyclin B1 but also by depleting Cdc14B or Cdh1. Notably, oocytes rescued by cyclin B1 overexpression exhibited normal spindle and chromosome organization with intact kinetochore-microtubule attachments. In addition, after being rescued by cyclin B1 overexpression, Mis12-depleted oocytes normally extruded polar bodies. Moreover, Mis12-depleted oocytes formed pronuclear structures after fertilization but failed to develop beyond zygotes. Interestingly, Mis12 was localized in the cytoplasm and spindle poles in oocytes, in contrast to kinetochore localization in somatic cells. Therefore, our results demonstrate that Mis12 is required for meiotic G2/M transition but is dispensable for meiotic progression through meiosis I and II.

Microtubules are necessary for proper Reticulon localization during mitosis.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

Spherical spindle shape promotes perpendicular cortical orientation by preventing isometric cortical pulling on both spindle poles during C. elegans female meiosis.

Meiotic spindles are positioned perpendicular to the oocyte cortex to facilitate segregation of chromosomes into a large egg and a tiny polar body. In C. elegans, spindles are initially ellipsoid and parallel to the cortex before shortening to a near-spherical shape with flattened poles and then rotating to the perpendicular orientation by dynein-driven cortical pulling. The mechanistic connection between spindle shape and rotation has remained elusive. Here, we have used three different genetic backgrounds to manipulate spindle shape without eliminating dynein-dependent movement or dynein localization. Ellipsoid spindles with flattened or pointed poles became trapped in either a diagonal or a parallel orientation. Mathematical models that recapitulated the shape dependence of rotation indicated that the lower viscous drag experienced by spherical spindles prevented recapture of the cortex by astral microtubules emanating from the pole pivoting away from the cortex. In addition, maximizing contact between pole dynein and cortical dynein stabilizes flattened poles in a perpendicular orientation, and spindle rigidity prevents spindle bending that can lock both poles at the cortex. Spindle shape can thus promote perpendicular orientation by three distinct mechanisms.