ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT‐stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral‐MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.     

The Abnormal Spindle-like,Microcephaly-associated (ASPM) Gene Encodes a Centrosomal Protein

Homozygous mutations in the abnormal spindle-like, microcephaly-associatedASPM gene are the leading cause of autosomal recessive primary microcephaly. ASPM isthe putative human ortholog of the Drosophila melanogaster abnormal spindles gene(asp), which is essential for mitotic spindle function. Here, we report thatdownregulation of endogenous ASPM by siRNA decreases protein levels of endogenousBRCA1. ASPM localizes to the centrosome in interphase and to the spindle poles fromprophase through telophase. These findings indicate that ASPM may be involved inmitotic spindle function, possibly, through regulation of BRCA1.     

Apoptotic regulators promote cytokinetic midbody degradation in C. elegans

Cell death genes are essential for apoptosis and other cellular events, but their nonapoptotic functions are not well understood. The midbody is an important cytokinetic structure required for daughter cell abscission, but its fate after cell division remains elusive in metazoans. In this paper, we show through live-imaging analysis that midbodies generated by Q cell divisions in Caenorhabditis elegans were released to the extracellular space after abscission and subsequently internalized and degraded by the phagocyte that digests apoptotic Q cell corpses. We further show that midbody degradation is defective in apoptotic cell engulfment mutants. Externalized phosphatidylserine (PS), an engulfment signal for corpse phagocytosis, exists on the outer surface of the midbody, and inhibiting PS signaling delayed midbody clearance. Thus, our findings uncover a novel function of cell death genes in midbody internalization and degradation after cell division.     

Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.     

The actin-capping protein CapG localizes to microtubule-dependent organelles during the cell cycle

Extensive cross-talk between the actin and the microtubule cytoskeletons has been reported. Especially in mitosis, processes dependent on actin- and microtubule-based structures alternate and regulate each other in a complex cascade leading to division into two daughter cells. Here, we have studied the subcellular localization of the filamentous actin-capping protein CapG. Fluorescence microscopy of endogenous CapG and EGFP-tagged CapG revealed CapG localization at the mother centriole in interphase, the mitotic spindle in mitosis and the midbody ring in abscission. Surprisingly, nucleoporin Nup62, an interaction partner of CapG, also localized to the midbody ring at the end of abscission and colocalized with CapG. We propose a role for the actin-binding protein CapG as a mediator of cross-talk between the actin cytoskeleton and microtubule-based organelles that regulate cell division.     

Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and cleavage stage embryos.     

Dynein LIC1 localizes to the mitotic spindle and midbody and LIC2 localizes to spindle poles during cell division

CD-1 (cytoplasmic dynein-1) is a multisubunit motor protein complex involved in intracellular trafficking and mitosis. The dynein LIC (light intermediate chain) subunits, LIC1 (DLIC-1, gene symbol DYNC1LI1) and LIC2 (DLIC-2, gene symbol DYNC1LI2), associate with the dynein HC (heavy chain) in a mutually exclusive manner and thus define distinct functional CD-1 complexes. Here, we analysed the mitotic distribution of LIC1 and LIC2. We found that from metaphase through anaphase, LIC1 localizes to the mitotic spindle and concentrates within the midbody during the abscission step of cytokinesis. Conversely, LIC2 strongly localizes to the spindle poles from prophase through telophase. These data suggest distinct functions for LIC1 and LIC2-containing CD-1 complexes during cell division.     

Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission

The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.     

Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle

Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.     

WDR62 localizes katanin at spindle poles to ensure synchronous chromosome segregation

Mutations in the WDR62 gene cause primary microcephaly, a pathological condition often associated with defective cell division that results in severe brain developmental defects. The precise function and localization of WDR62 within the mitotic spindle is, however, still under debate, as it has been proposed to act either at centrosomes or on the mitotic spindle. Here we explored the cellular functions of WDR62 in human epithelial cell lines using both short-term siRNA protein depletions and long-term CRISPR/Cas9 gene knockouts. We demonstrate that WDR62 localizes at spindle poles, promoting the recruitment of the microtubule-severing enzyme katanin. Depletion or loss of WDR62 stabilizes spindle microtubules due to insufficient microtubule minus-end depolymerization but does not affect plus-end microtubule dynamics. During chromosome segregation, WDR62 and katanin promote efficient poleward microtubule flux and favor the synchronicity of poleward movements in anaphase to prevent lagging chromosomes. We speculate that these lagging chromosomes might be linked to developmental defects in primary microcephaly.     

Nuclear localization of C-terminal domains of the kinesin-like protein MKLP-1.

The successful execution of mitosis in mammalian cells requires the activities of numerous kinesin-like proteins. The Mitotic Kinesin-Like Protein-1 (MKLP-1) localizes to the spindle equator and is believed to participate in the separation of spindle poles during anaphase B. Injection of antibodies against MKLP-1 into dividing cells results in cell cycle arrest, suggesting that MKLP-1 is essential for mitosis. To further characterize MKLP-1, constructs encoding C-terminal domains of MKLP-1 were expressed as fusions to the green fluorescent protein and localized in HeLa cells. All constructs localized to the nucleus indicating the presence of at least one nuclear localization sequence in the C-terminus of the protein. C-terminal domains of MKLP-1 expressed in insect cells also localized to the nucleus as shown by subcellular fractionation. These proteins remained tightly associated with the nucleus following both detergent and salt extraction, suggesting a tight interaction with a component of the nucleus.     

Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis

TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.     

The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring–derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.     

ALIX and ESCRT-III Coordinately Control Cytokinetic Abscission during Germline Stem Cell Division In Vivo

Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.     

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

The ciliopathy-linked protein CCDC66 is only known for its ciliary functions. This study reveals that CCDC66 also has extensive non-ciliary functions, localizing to the spindle poles, spindle midzone, central spindle and midbody throughout cell division, where it regulates mitosis and cytokinesis by promoting microtubule nucleation and organization.     

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.     

Midbody localization of vinexin recruits rhotekin to facilitate cytokinetic abscission

Vinexin is a SH3 domain-containing adaptor protein that has diverse roles in cell adhesion, signal transduction, gene regulation and stress granule assembly. In this study, we found that vinexin localizes at the midbody during cell division and facilitates cytokinesis. Knockdown of vinexin in HeLa cells delayed the mitotic cell cycle progression and increased the time of cell abscission and the failure to resolve the cytoplasmic bridge. Midbody-localized vinexin is essential for recruiting rhotekin to this structure for cytokinesis because overexpression of a vinexin mutant without a rhotekin-binding motif or knockdown of rhotekin also impaired cytokinetic abscission and increased the number of cells arrested at the midbody stage. Aberrant expression of vinexin and rhotekin in various cancers has been implicated to promote metastasis because of their functions in cell adhesion and signaling. Our findings reveal a novel role of vinexin and rhotekin in cytokinetic abscission and provide another perspective of how both molecules may affect oncogenic transformation via this fundamental cell cycle process.     

GM130, a cis-Golgi protein,regulates meiotic spindle assembly and asymmetric division in mouse oocyte

GM130, a cis-Golgi protein, plays key roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that GM130 was localized to the spindle poles at both metaphase I and metaphase II stages and associated with the midbody at telophase I stage. The association of GM130 with spindle poles was further confirmed by its colocalization with the centrosome-associated proteins, MEK1/2. By nocodazole treatment, we clarified that GM130 localization was consistently dependent on spindle assembly. Then we investigated the possible function of GM130 by specific morpholino microinjection. This treatment caused abnormal spindle formation, and decreased first polar body extrusion. Our results showed that knockdown of GM130 impaired the localization of MTOCs proteins γ-tubulin and Plk1. Using live cell imaging we observed that depletion of GM130 affected spindle migration and resulted in elongated spindle and large polar body extrusion. We further found that depletion of GM130 blocked p-MEK1/2 accumulation at the spindle poles. And, it was shown that GM130 detached from the spindle poles in oocytes treated with MEK specific inhibitor U0126. Taken together, our results suggested that GM130 regulates microtubule organization and might cooperate with the MAPK pathway to play roles in spindle organization, migration and asymmetric division during mouse oocyte maturation.