Annexin A2 is required for the early steps of cytokinesis

Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium- and phospholipid-binding protein implicated in cortical actin remodeling. We report that annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In the absence of annexin A2, the small GTPase RhoA—which regulates cortical cytoskeletal rearrangement—fails to form a compact ring at the equatorial plane. Furthermore, annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in central spindle–equatorial plasma membrane communication.     

The class I PITP giotto is required for Drosophila cytokinesis

Phosphatidylinositol transfer proteins (PITPs) are highly conserved polypeptides that bind phosphatidylinositol or phosphatidylcholine monomers, facilitating their transfer from one membrane compartment to another . Although PITPs have been implicated in a variety of cellular functions, including lipid-mediated signaling and membrane trafficking, the precise biological roles of most PITPs remain to be elucidated . Here we show for the first time that a class I PITP is involved in cytokinesis. We found that giotto (gio), a Drosophila gene that encodes a class I PITP, serves an essential function required for both mitotic and meiotic cytokinesis. Neuroblasts and spermatocytes from gio mutants both assemble regular actomyosin rings. However, these rings fail to constrict to completion, leading to cytokinesis failures. Moreover, gio mutations cause an abnormal accumulation of Golgi-derived vesicles at the equator of spermatocyte telophases, suggesting that Gio is implicated in membrane-vesicle fusion. Consistent with these results, we found that Gio is enriched at the cleavage furrow, the ER, and the spindle envelope. We propose that Gio mediates transfer of lipid monomers from the ER to the equatorial membrane, causing a specific local enrichment in phosphatidylinositol. This change in membrane composition would ultimately facilitate vesicle fusion, allowing membrane addition to the furrow and/or targeted delivery of proteins required for cytokinesis.     

Discs large 5 is required for polarization of citron kinase in mitotic neural precursors

Citron kinase (CitK), a protein essential to neurogenic cell division in the central nervous system, is highly polarized in neural progenitors. The mechanisms that polarize CitK to cellular domains that line the ventricular surface of neuroepithelium are currently not known. Here we report that Discs large 5 (Dlg5), a member of the MAGUK family, is an interactor of CitK required for CitK polarization. The CitK-Dlg5 interaction was first revealed in a protein array screen of proteins containing PDZ domains, and then subsequently confirmed by co-immunoprecipitation. Moreover, in Dlg5^-/- mice CitK fails to polarize in mitotic neuronal precursors. In addition, the total number of mitotic progenitors and the ratio of ventricular to abventricular mitotic progenitors in developing neocortex are significantly decreased in Dlg5^-/- embryos. Dlg5 is therefore required to maintain the polarization of a protein essential to neurogenic cytokinesis, and plays a role in localizing cell divisions to the surface of the lateral ventricles in embryonic brain.     

The kinase activity of human Rio1 is required for final steps of cytoplasmic maturation of 40S subunits

RIO proteins form a conserved family of atypical protein kinases. Humans possess three distinct RIO kinases-hRio1, hRio2, and hRio3, of which only hRio2 has been characterized with respect to its role in ribosomal biogenesis. Here we show that both hRio1 and hRio3, like hRio2, are associated with precursors of 40S ribosomal subunits in human cells. Furthermore, we demonstrate that depletion of hRio1 by RNA interference affects the last step of 18S rRNA maturation and causes defects in the recycling of several trans-acting factors (hEnp1, hRio2, hLtv1, hDim2/PNO1, and hNob1) from pre-40S subunits in the cytoplasm. Although the effects of hRio1 and hRio2 depletion are similar, we show that the two kinases are not fully interchangeable. Moreover, rescue experiments with a kinase-dead mutant of hRio1 revealed that the kinase activity of hRio1 is essential for the recycling of the endonuclease hNob1 and its binding partner hDim2 from cytoplasmic pre-40S. Kinase-dead hRio1 is trapped on pre-40S particles containing hDim2 and hNob1 but devoid of hEnp1, hLtv1, and hRio2. These data reveal a role of hRio1 in the final stages of cytoplasmic pre-40S maturation.     

Phosphorylation of Ataxin-10 by polo-like kinase 1 is required for cytokinesis

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder,whose symptoms include cerebellar ataxia and epilepsy. The disease is caused by ATTCT expansion in the ATXN10 gene, which encodes the Ataxin-10 protein. Here we identified polo-like kinase 1 (Plk1) as one of Ataxin-10's binding partners. We show that epitope-tagged Ataxin-10 and Plk1 coimmunoprecipitate, and Plk1 phosphorylates Ataxin-10 at S77 and T82 in vitro. Knockdown of ATXN10 with siRNA in HeLa cells results in cytokinesis defects-multinucleation, which are rescued by wild-type Ataxin-10, but not the phosphor-deficient 2A mutant. Phosphorylation-specific antibodies towards pS77 detect specific signals at the midbody. Like the knockdown, overexpression of the 2A mutant generates multinucleated cells and the 2A mutant shows decreased interaction with the Plk1 polo-box domain. In addition, we found that Ataxin-10 is ubiquitinated, and is subject to proteasome-dependent degradation, which is delayed in the 2A mutant. We propose a model in which Plk1 phosphorylation of Ataxin-10 influences its degradation and cytokinesis, which may provide mechanistic insight to SCA10's pathogenesis.     

Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.     

KIF14 and citron kinase act together to promote efficient cytokinesis

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.     

Two-pore channel 1 interacts with citron kinase,regulating completion of cytokinesis

Two-pore channels (TPC1, 2, and 3) are recently identified endolysosmal ion channels, but remain poorly characterized. In this study, we show for the first time a role for TPC1 in cytokinesis, the final step in cell division. HEK 293 T-REx cells inducibly overexpressing TPC1 demonstrated a lack of proliferation accompanied by multinucleation and an increase in G2/M cycling cells. Increased TPC1 was associated with a concomitant accumulation of active RhoGTP and a decrease in phosphorylated myosin light chain (MLC). Finally, we demonstrated a novel interaction between TPC1 and citron kinase (CIT). These results identify TPC1 as a central component of cytokinetic control, specifically during abscission, and introduce a means by which the endolysosomal system may play an active role in this process.     

Two-pore channel 1 interacts with citron kinase,regulating completion of cytokinesis

Two-pore channels (TPC1, 2, and 3) are recently identified endolysosmal ion channels, but remain poorly characterized. In this study, we show for the first time a role for TPC1 in cytokinesis, the final step in cell division. HEK 293 T-REx cells inducibly overexpressing TPC1 demonstrated a lack of proliferation accompanied by multinucleation and an increase in G2/M cycling cells. Increased TPC1 was associated with a concomitant accumulation of active RhoGTP and a decrease in phosphorylated myosin light chain (MLC). Finally, we demonstrated a novel interaction between TPC1 and citron kinase (CIT). These results identify TPC1 as a central component of cytokinetic control, specifically during abscission, and introduce a means by which the endolysosomal system may play an active role in this process.     

Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.     

Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.     

Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.     

Nessun Dorma, a novel centralspindlin partner, is required for cytokinesis in Drosophila spermatocytes

Cytokinesis, the final step of cell division, usually ends with the abscission of the two daughter cells. In some tissues, however, daughter cells never completely separate and remain interconnected by intercellular bridges or ring canals. In this paper, we report the identification and analysis of a novel ring canal component, Nessun Dorma (Nesd), isolated as an evolutionarily conserved partner of the centralspindlin complex, a key regulator of cytokinesis. Nesd contains a pectin lyase-like domain found in proteins that bind to polysaccharides, and we present evidence that it has high affinity for β-galactosides in vitro. Moreover, nesd is an essential gene in Drosophila melanogaster, in which it is required for completion of cytokinesis during male meiosis and possibly in female germline cells. Our findings indicate that Nesd is a novel carbohydrate-binding protein that functions together with centralspindlin in late cytokinesis, thus highlighting the importance of glycosylation in this process.     

A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.     

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.     

RalA-exocyst-dependent recycling endosome trafficking is required for the completion of cytokinesis

In eukaryotic cells, recycling endosome-mediated trafficking contributes to the completion of cytokinesis, in a manner under the control of the centrosome. We report that the exocyst complex and its interacting GTPase RalA play a critical role in this polarized trafficking process. RalA resides in the recycling endosome and relocates from the pericentrosomal region to key cytokinetic structures including the cleavage furrow, and later, the abscission site. This event is coupled to the dynamic redistribution of the exocyst proteins. These associate with the centrosome in interphase and concentrate on the central spindle/midbody during cytokinesis. Disruption of RalA-exocyst function leads to cytokinesis failure in late stages, particularly abscission, resembling the cytokinesis defects induced by loss of centrosome function. These data suggest that RalA and the exocyst may regulate vesicle delivery to the centrosome-related abscission site during the terminal stage of cytokinesis, implicating RalA as a critical regulator of cell cycle progression.     

Rab11 is required for membrane trafficking and actomyosin ring constriction in meiotic cytokinesis of Drosophila males

Rab11 is a small GTPase that regulates several aspects of vesicular trafficking. Here, we show that Rab11 accumulates at the cleavage furrow of Drosophila spermatocytes and that it is essential for cytokinesis. Mutant spermatocytes form regular actomyosin rings, but these rings fail to constrict to completion, leading to cytokinesis failures. rab11 spermatocytes also exhibit an abnormal accumulation of Golgi-derived vesicles at the telophase equator, suggesting a defect in membrane-vesicle fusion. These cytokinesis phenotypes are identical to those elicited by mutations in giotto (gio) and four wheel drive (fwd) that encode a phosphatidylinositol transfer protein and a phosphatidylinositol 4-kinase, respectively. Double mutant analysis and immunostaining for Gio and Rab11 indicated that gio, fwd, and rab11 function in the same cytokinetic pathway, with Gio and Fwd acting upstream of Rab11. We propose that Gio and Fwd mediate Rab11 recruitment at the cleavage furrow and that Rab11 facilitates targeted membrane delivery to the advancing furrow.