Clathrin heavy chain mediates TACC3 targeting to mitotic spindles to ensure spindle stability

Mitotic spindles play essential roles in chromosome congression and segregation during mitosis. Aurora A regulates spindle assembly in part via phosphorylating human TACC3 on S558, which triggers TACC3 relocalization to mitotic spindles and stabilizes microtubules (MTs). In this study, we identified clathrin heavy chain (CHC) as an adaptor protein to recruit S558-phosphorylated TACC3 onto the spindle during mitosis for MT stabilization. CHC binds phospho-S558 TACC3 via its linker domain and first CHC repeat. CHC depletion or mutation on phospho-TACC3 binding abrogates TACC3 spindle relocalization. Depletion of either or both CHC and TACC3 yields similar defective phenotypes: loss of ch-TOG on spindles, disorganized spindles, and chromosome misalignment with comparable mitotic delay. Our findings elucidate the association between aurora A phosphorylation and spindle apparatus and demonstrate that regulation from aurora A is mediated by CHC in recruiting phospho-TACC3 and subsequently ch-TOG to mitotic spindles.     

A TACC3/ch-TOG/clathrin complex stabilises kinetochore fibres by inter-microtubule bridging

Kinetochore fibres (K-fibres) of the spindle apparatus move chromosomes during mitosis. These fibres are discrete bundles of parallel microtubules (MTs) that are crosslinked by inter-MT 'bridges' that are thought to improve fibre stability during chromosomal movement. The identity of these bridges is unknown. Clathrin is a multimeric protein that has been shown to stabilise K-fibres during early mitosis by a mechanism independent of its role in membrane trafficking. In this study, we show that clathrin at the mitotic spindle is in a transforming acidic colied-coil protein 3 (TACC3)/colonic, hepatic tumour overexpressed gene (ch-TOG)/clathrin complex. The complex is anchored to the spindle by TACC3 and ch-TOG. Ultrastructural analysis of clathrin-depleted K-fibres revealed a selective loss of a population of short inter-MT bridges and a general loss of MTs. A similar loss of short inter-MT bridges was observed in TACC3-depleted K-fibres. Finally, immunogold labelling confirmed that inter-MT bridges in K-fibres contain clathrin. Our results suggest that the TACC3/ch-TOG/clathrin complex is an inter-MT bridge that stabilises K-fibres by physical crosslinking and by reducing rates of MT catastrophe.     

Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

Acomplex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3–clathrin binding. A dileucine motif and Aurora A–phosphorylated serine 558 on TACC3 bound to the “ankle” of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3–clathrin.     

The clathrin heavy chain isoform CHC22 functions in a novel endosomal sorting step

Clathrin heavy chain 22 (CHC22) is an isoform of the well-characterized CHC17 clathrin heavy chain, a coat component of vesicles that mediate endocytosis and organelle biogenesis. CHC22 has a distinct role from CHC17 in trafficking glucose transporter 4 (GLUT4) in skeletal muscle and fat, though its transfection into HEK293 cells suggests functional redundancy. Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells. CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer. In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome–TGN transport as critical for GLUT4 trafficking. Like CHC22, syntaxin 10 is not expressed in mice but is present in humans and other vertebrates, implicating two species-restricted endosomal traffic proteins in GLUT4 transport.     

Asymmetric localization of the CDC25B phosphatase to the mother centrosome during interphase

The Cyclin-Dependent Kinase (CDK)-activating phosphatase CDC25B, localises to the centrosomes where its activity is both positively and negatively regulated by several kinases including Aurora A and CHK1. Our recent data also demonstrate a role for CDC25B in the centrosome duplication cycle and microtubule nucleation in interphase that appears to involve the recruitment of γ-tubulin to the centrosomes. In the present study, we report that CDC25B, along with CHK1, CDK1 and WEE1, localise asymmetrically around the mother centrosome from S to G2-phases, and gradually become evenly distributed to the two centrosomes by late G2 phase, concomitant with centrosome maturation. We further demonstrate that siRNA inhibition of CDC25B results in an accumulation of cells in G2 phase with two separated centrosomes, each containing only a single centriole, suggesting a requirement for CDC25B in centriole duplication. We propose that the localisation of key cell cycle regulators to the mother centrosome ensures synchrony between the centrosome duplication and cell division cycles.     

Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.     

A microtubule-independent role for centrosomes and aurora a in nuclear envelope breakdown

Aurora A kinase localizes to centrosomes and is required for centrosome maturation and spindle assembly. Here we describe a microtubule-independent role for Aurora A and centrosomes in nuclear envelope breakdown (NEBD) during the first mitotic division of the C. elegans embryo. Aurora A depletion does not alter the onset or kinetics of chromosome condensation, but dramatically lengthens the interval between the completion of condensation and NEBD. Inhibiting centrosome assembly by other means also lengthens this interval, albeit to a lesser extent than Aurora A depletion. By contrast, centrosomally nucleated microtubules and the nuclear envelope-associated motor dynein are not required for timely NEBD. These results indicate that mitotic centrosomes generate a diffusible factor, which we propose is activated Aurora A, that promotes NEBD. A positive feedback loop, in which an Aurora A-dependent increase in centrosome size promotes Aurora A activation, may temporally couple centrosome maturation to NEBD during mitotic entry.     

Centrosomes at M phase act as a scaffold for the accumulation of intracellular ubiquitinated proteins

Centrosome size varies considerably during the cell cycle; it is greatest during metaphase, partly because of pericentriolar matrix recruitment and an increase in microtubule-organizing activity. However, the mechanism of centrosome maturation during M phase is poorly defined. In the present study, we identified and quantified centrosomal proteins during S and M phases using stable isotope labeling by amino acids in cell culture (SILAC) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS). We identified 991 proteins, of which 310 and 325 proteins were upregulated during S and M phases, respectively. Ubiquitinated proteins containing K48- and K63-linked polyubiquitin chains accumulated in the centrosomes during M phase, although 26S proteasome activity in the centrosomes did not markedly differ between S and M phases. Conversely, cytoplasmic dynein, which transports ubiquitinated proteins to the centrosomes, increased 2-fold in the centrosomes during M phase relative to S phase. Furthermore, PYR-41, a ubiquitin E1 inhibitor, reduced centrosome size during metaphase, causing increased aneuploidy. RNA interference suppression of Ecm29, which inhibits proteasome activity, decreased the accumulation of ubiquitinated proteins in the centrosomes. These results show that accumulation of ubiquitinated proteins promotes centrosome maturation during M phase and further suggest a novel function of centrosomes as a scaffold temporarily gathering intracellular ubiquitinated proteins.     

Phosphorylation of histone H3 serine 10 in early mouse embryos: Active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses

Cell division in mammalian cells is regulated by Aurora kinases. The activity of Aurora A is indispensable for correct function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation. Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase. Data concerning the Aurora B activity and H3S10Ph in embryonic cells are limited to primordial and maturing oocytes and advanced pronuclei in zygotes. In the present study we have analyzed H3S10Ph in 1- and 2-cell mouse embryos. We show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and subsequently phosphorylated de novo at late S phase of the first and second cell cycle. These results show that the H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. The behaviour of thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible for de novo H3S10 phosphorylation, most probably Aurora B, are active until G1 of the first cell cycle of mouse embryo. The inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle checkpoint remained active.     

Inhibition of centrosome protein assembly leads to p53-dependent exit from the cell cycle

Previous evidence has indicated that an intact centrosome is essential for cell cycle progress and that elimination of the centrosome or depletion of individual centrosome proteins prevents the entry into S phase. To investigate the molecular mechanisms of centrosome-dependent cell cycle progress, we performed RNA silencing experiments of two centrosome-associated proteins, pericentriolar material 1 (PCM-1) and pericentrin, in primary human fibroblasts. We found that cells depleted of PCM-1 or pericentrin show lower levels of markers for S phase and cell proliferation, including cyclin A, Ki-67, proliferating cell nuclear antigen, minichromosome maintenance deficient 3, and phosphorylated retinoblastoma protein. Also, the percentage of cells undergoing DNA replication was reduced by >50%. At the same time, levels of p53 and p21 increased in these cells, and cells were predisposed to undergo senescence. Conversely, depletion of centrosome proteins in cells lacking p53 did not cause any cell cycle arrest. Inhibition of p38 mitogen-activated protein kinase rescued cell cycle activity after centrosome protein depletion, indicating that p53 is activated by the p38 stress pathway.     

Plk1 regulates mitotic Aurora A function through βTrCP-dependent degradation of hBora

Polo-like kinase 1 (Plk1) and Aurora A play key roles in centrosome maturation, spindle assembly, and chromosome segregation during cell division. Here we show that the functions of these kinases during early mitosis are coordinated through Bora, a partner of Aurora A first identified in Drosophila. Depletion of human Bora (hBora) results in spindle defects, accompanied by increased spindle recruitment of Aurora A and its partner TPX2. Conversely, hBora overexpression induces mislocalization of Aurora A and monopolar spindle formation, reminiscent of the phenotype seen in Plk1-depleted cells. Indeed, Plk1 regulates hBora. Following Cdk1-dependent recruitment, Plk1 triggers hBora destruction by phosphorylating a recognition site for [Formula: see text]. Plk1 depletion or inhibition results in a massive accumulation of hBora, concomitant with displacement of Aurora A from spindle poles and impaired centrosome maturation, but remarkably, co-depletion of hBora partially restores Aurora A localization and bipolar spindle formation. This suggests that Plk1 controls Aurora A localization and function by regulating cellular levels of hBora.     

Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89

Aurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA.     

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.     

Beyond focal adhesions: Integrin linked kinase associates with tubulin and regulates mitotic spindle organization

Integrin Linked kinase (ILK) is a member of a multiprotein complex at focal adhesions which interacts with actin. Here, it functions as a kinase and adapter protein to regulate diverse cellular processes. Gene knockout studies have demonstrated critical roles for ILK in embryonic development and in organ and tissue homeostasis. However, ILK is overexpressed in many human cancers and experimental overexpression in non-transformed cells results in the acquisition of several oncogenic phenotypes.Proteomic based approaches to identify ILK binding partners have now identified tubulins and many centrosomal and mitotic spindle associated proteins as ILK interactors in addition to the expected focal adhesion, actin interacting, proteins. Further analysis has shown that ILK co-localizes with several of these proteins to the centrosome and inhibition or depletion of ILK causes mitotic spindle defects by disrupting Aurora A kinase/TACC3/ch-TOG interactions. Here we discuss the finding that ILK is a member of a tubulin-based multiprotein complex at the centrosome, whether this may interact with the focal adhesion pool of ILK, and identify potential mechanisms by which ILK regulates the organization of the mitotic spindle. We also discuss the implications of ILK’s mitotic role for cancer progression and highlight the potential use of ILK inhibitors as novel anti-mitotic chemotherapeutics.     

Depletion of Aurora A leads to upregulation of FoxO1 to induce cell cycle arrest in hepatocellular carcinoma cells

Aurora A kinase has drawn considerable attention as a therapeutic target for cancer therapy. However, the underlying molecular and cellular mechanisms of the anticancer effects of Aurora A kinase inhibition are still not fully understood. Herein, we show that depletion of Aurora A kinase by RNA interference (RNAi) in hepatocellular carcinoma (HCC) cells upregulated FoxO1 in a p53-dependent manner, which induces cell cycle arrest. Introduction of an RNAi-resistant Aurora A kinase into Aurora A-knockdown cells resulted in downregulation of FoxO1 expression and rescued proliferation. In addition, silencing of FoxO1 in Aurora A-knockdown cells allowed the cells to exit cytostatic arrest, which, in turn, led to massive cell death. Our results suggest that FoxO1 is responsible for growth arrest at the G₂/M phase that is induced by Aurora A kinase inhibition.     

Condensation-decondensation of the gamma-tubulin containing material in the absence of a structurally visible organelle during the cell cycle of Physarum plasmodia.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.     

The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation

Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC), consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and cytokinetic initiation. This is the first time to our knowledge that the dual functions of an Aurora B homolog is dissected and separated into two clearly distinct time frames in a cell cycle.