Spindle Pole Microtubules Research Articles

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.

Loss of kinesin-14 results in aneuploidy via kinesin-5-dependent microtubule protrusions leading to chromosome cut.

Aneuploidy – chromosome instability leading to incorrect chromosome number in dividing cells – can arise from defects in centrosome duplication, bipolar spindle formation, kinetochore-microtubule attachment, chromatid cohesion, mitotic checkpoint monitoring, or cytokinesis. As most tumors show some degree of aneuploidy, mechanistic understanding of these pathways has been an intense area of research to provide potential therapeutics. Here, we present a mechanism for aneuploidy in fission yeast based on spindle pole microtubule defocusing by loss of kinesin-14 Pkl1, leading to kinesin-5 Cut7-dependent aberrant long spindle microtubule minus end protrusions that push the properly segregated chromosomes to the site of cell division, resulting in chromosome cut at cytokinesis. Pkl1 localization and function at the spindle pole is mutually dependent on spindle pole-associated protein Msd1. This mechanism of aneuploidy bypasses the known spindle assembly checkpoint that monitors chromosome segregation.

EDD, a Ubiquitin-protein Ligase of the N-end Rule Pathway, Associates with Spindle Assembly Checkpoint Components and Regulates the Mitotic Response to Nocodazole

In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G2/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis.

Class-1 Myosins at the Interphase between Spindles and Membranes

Class-1 myosins play key roles in the dynamic organization of the cytoskeleton and membrane shape as they act as divalent cross-linkers interconnecting and generating force between both actin and membrane systems. Previously we have shown that the long-tailed Dictyostelium myosin-1C not only is involved in membrane remodeling during endocytic processes, but also plays a role in spindle dynamics during mitosis through direct microtubule interactions. Here, we characterized myosin-1D as the second myosin-1 member with roles in mitosis. Our studies reveal that myosin-1D binds spindle pole microtubules and associates with the nuclear envelope of dividing cells. Myosin-1D targets microtubules and nuclear structures early during mitosis and decorates the membrane embedded chromatin masses until the end of cell division. This suggests a role of the motor in nuclear membrane fission by mediating microtubule-membrane interactions. Since the nuclear envelope in Dictyostelium remains intact throughout mitosis, except for regions of perforations that are found near the spindle poles, myosin-1D appears to act as a dynamic linker connecting spindle pole microtubules to the nuclear membrane important to hold the envelope together as it gets stretched upon spindle elongation. To investigate the mechanisms that govern myosin-1s to distinct mitotic structures, we have generated a set of motor and tail domain constructs and characterized binding affinities and kinetics of intermolecular interactions with lipid membranes, actin, and microtubules. Our results show that differences in strength and specificity of lipid and microtubule binding and regulatory roles of the tails account for the specialized functions of the motors in mitosis.

Shedding Light on Mysterious Microtubules

We have known for decades that vertebrate kinetochores can nucleate microtubules. However, the role of such microtubules was unclear. Kitamura et al. investigated this issue by following a marked budding yeast centromere after its conditional reactivation to examine microtubule capture at unattached kinetochores. They found not only that yeast kinetochores can nucleate microtubules, but also that such microtubules facilitate attachment by decreasing the time required for spindle-pole-associated microtubules to make contact with unattached kinetochores. Historically, the fact that microtubules nucleated at kinetochores are opposite in polarity to those in mature spindle fibers was used to argue that they were not physiologically relevant for spindle assembly. Kitamura et al. have now shown that they, in fact, do play a role, and then rapidly depolymerize after spindle fiber attachments form. In sum, this paper provides a function for a previously mysterious microtubule population and outlines a surprising and dynamic mechanism through which kinetochore-originated microtubules assist spindle pole microtubules to efficiently locate unattached kinetochores. It also shows how this assistance mechanism is shut off to dim the locator beacon after correct attachments have been made. Understanding Kinetochore-Nucleated Microtubules Tomoyuki Tanaka, with the help of Etsushi Kitamura and Shinya Komoto, explains his group's key findings regarding the roles of kinetochore-generated microtubules in mitosis.

Kinetochore–microtubule interactions: steps towards bi-orientation

Eukaryotic cells segregate their chromosomes accurately to opposite poles during mitosis, which is necessary for maintenance of their genetic integrity. This process mainly relies on the forces generated by kinetochore-microtubule (KT-MT) attachment. During prometaphase, the KT initially interacts with a single MT extending from a spindle pole and then moves towards a spindle pole. Subsequently, MTs from the other spindle pole also interact with the KT. Eventually, one sister KT becomes attached to MTs from one pole while the other sister to those from the other pole (sister KT bi-orientation). If sister KTs interact with MTs with aberrant orientation, this must be corrected to attain proper bi-orientation (error correction) before the anaphase is initiated. Here, I discuss how KTs initially interact with MTs and how this interaction develops into bi-orientation; both processes are fundamentally crucial for proper chromosome segregation in the subsequent anaphase.

Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis

SummaryIn early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores. However, we now have evidence that, during early mitosis in budding yeast, microtubules are generated at kinetochores with distal plus ends. These kinetochore-derived microtubules interact along their length with microtubules that extend from a spindle pole, facilitating kinetochore loading onto the lateral surface of spindle pole microtubules. Once kinetochores are loaded, microtubules are no longer generated at kinetochores, and those that remain disappear rapidly and do not contribute to the metaphase spindle. Stu2 (the ortholog of vertebrate XMAP215/ch-TOG) localizes to kinetochores and plays a central role in regulating kinetochore-derived microtubules. Our work provides insight into microtubule generation at kinetochores and the mechanisms that facilitate initial kinetochore interaction with spindle pole microtubules.

Protein complexes at the microtubule organizing center regulate bipolar spindle assembly

Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G1/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22ts, encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22ts we found that stabilized microtubules or increased bundling can rescue cut7-22ts associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on γ-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to γ-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/γ-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22ts strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.

Distinct Dgrip84 Isoforms Correlate with Distinct γ-Tubulins inDrosophila

Gamma-tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, gamma-tubulin exists in a multiprotein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). gamma-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the gamma-tubulin ring complex (gammaTuRC). Curiously, many organisms (including humans) have two distinct gamma-tubulin genes. In Drosophila, where the two gamma-tubulin isotypes have been studied most extensively, the gamma-tubulin genes are developmentally regulated: the "maternal" gamma-tubulin isotype (named gammaTub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second gamma-tubulin isotype (gammaTub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both gamma-tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes: gammaTub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of gammaTub23C at the centrosome increases at the beginning of mitosis, and gammaTub23C also associates with spindle pole microtubules. Here, we show that gammaTub23C forms discrete complexes that closely resemble the complexes formed by gammaTub37CD. Surprisingly, however, gammaTub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the gamma-tubulin complexes formed with gammaTub37CD and gammaTub23C.

TACC1-chTOG-Aurora A protein complex in breast cancer.

The three human TACC (transforming acidic coiled-coil) genes encode a family of proteins with poorly defined functions that are suspected to play a role in oncogenesis. A Xenopus TACC homolog called Maskin is involved in translational control, while Drosophila D-TACC interacts with the microtubule-associated protein MSPS (Mini SPindleS) to ensure proper dynamics of spindle pole microtubules during cell division. We have delineated here the interactions of TACC1 with four proteins, namely the microtubule-associated chTOG (colonic and hepatic tumor-overexpressed gene) protein (ortholog of Drosophila MSPS), the adaptor protein TRAP (tudor repeat associator with PCTAIRE2), the mitotic serine/threonine kinase Aurora A and the mRNA regulator LSM7 (Like-Sm protein 7). To measure the relevance of the TACC1-associated complex in human cancer we have examined the expression of the three TACC, chTOG and Aurora A in breast cancer using immunohistochemistry on tissue microarrays. We show that expressions of TACC1, TACC2, TACC3 and Aurora A are significantly correlated and downregulated in a subset of breast tumors. Using siRNAs, we further show that depletion of chTOG and, to a lesser extent of TACC1, perturbates cell division. We propose that TACC proteins, which we also named 'Taxins', control mRNA translation and cell division in conjunction with microtubule organization and in association with chTOG and Aurora A, and that these complexes and cell processes may be affected during mammary gland oncogenesis.

Localization of Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle.

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.

Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action.

Microtubules are highly dynamic cellular polymers made of alphabeta-tubulin and associated proteins. They play a key role during mitosis, participating in the exact organization and function of the spindle, and are critical for assuring the integrity of the segregated DNA. Therefore, they represent one of the more effective targets in current cancer therapy. Paclitaxel (Taxol) is the prototype of the taxane family of antitumor drugs, and it was the first natural product shown to stabilize microtubules. This unique mechanism of action is in contrast to other microtubule poisons, such as Vinca alkaloids, colchicine, and cryptophycines, which inhibit tubulin polymerization. Taxanes block cell cycle progression through centrosomal impairment, induction of abnormal spindles and suppression of spindle microtubule dynamics. Triggering of apoptosis by aberrant mitosis or by subsequent multinucleated G1-like state related to mitotic slippage, depends on cell type and drug schedule. The development of fluorescent derivatives of paclitaxel led us to locate spindle pole microtubules and centrosomes as main sub-cellular targets of cytotoxic taxoids in living cells. In this review we discuss these findings in the context of a cell cycle-dependent response to taxanes, based on the cellular targets, and the status of the implicated cell cycle checkpoints. We also review those events that can influence this response, like the different signal transduction pathways activated/inactivated in relation to Bcl-2 phosphorylation and induction of apoptosis, and the controversial role of the p53 status on cell sensitivity to paclitaxel. Finally, cell cycle-dependent resistance, an emerging concept in combination sequential chemotherapy, is discussed on the basis of the cell cycle-dependent mechanisms of action of taxanes.

Xklp2, a Novel Xenopus Centrosomal Kinesin-like Protein Required for Centrosome Separation during Mitosis

We describe a novel Xenopus plus end–directed kinesin-like protein (KLP), Xklp2, localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase. Using mitotic spindles assembled in Xenopus egg extracts and different recombinant GST–Xklp2 mutants, we show that this motor is targeted to spindle poles through its C-terminal domain. Xklp2-truncated polypeptides lacking the motor domain block centrosome separation and disrupt preassembled metaphase spindles. Antibodies directed against the tail of Xklp2 have a similar effect. These results show that Xklp2 protein is required for centrosome separation and maintenance of spindle bipolarity. This study is an example of the application of the dominant negative mutant effect on spindle assembly in Xenopus egg extracts, demonstrating the usefulness of this approach in probing the function of proteins in this system.