SHCBP1 is required for midbody organization and cytokinesis completion

The centralspindlin complex, which is composed of MKLP1 and MgcRacGAP, is one of the crucial factors involved in cytokinesis initiation. Centralspindlin is localized at the middle of the central spindle during anaphase and then concentrates at the midbody to control abscission. A number of proteins that associate with centralspindlin have been identified. These associating factors regulate furrowing and abscission in coordination with centralspindlin. A recent study identified a novel centralspindlin partner, called Nessun Dorma, which is essential for germ cell cytokinesis in Drosophila melanogaster. SHCBP1 is a human ortholog of Nessun Dorma that associates with human centralspindlin. In this report, we analyzed the interaction of SHCBP1 with centralspindlin in detail and determined the regions that are required for the interaction. In addition, we demonstrate that the central region is necessary for the SHCBP1 dimerization. Both MgcRacGAP and MKLP1 are degraded once cells exit mitosis. Similarly, endogenous and exogenous SHCBP1 were degraded with mitosis progression. Interestingly, SHCBP1 expression was significantly reduced in the absence of centralspindlin, whereas centralspindlin expression was not affected by SHCBP1 knockdown. Finally, we demonstrate that SHCBP1 depletion promotes midbody structure disruption and inhibits abscission, a final stage of cytokinesis. Our study gives novel insight into the role of SHCBP in cytokinesis completion.     

Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis

Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper our ability to test this hypothesis. We used loss- and gain-of-function approaches to examine the role of Diaphanous in Drosophila morphogenesis. We found that Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. We used constitutively active Diaphanous to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. We tested this hypothesis by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis.     

Down syndrome cell adhesion molecule is important for early development in Xenopus tropicalis

The Down syndrome cell adhesion molecule (DSCAM) is an Ig containing cell adhesion molecule with remarkable structural conservation throughout metazoans. In insects, DSCAM has 38,000 potential isoforms that convey axon guidance, fasciculation, and dendrite morphogenesis during neurodevelopment. In vertebrates, DSCAM is expressed throughout the nervous system and seems to also mediate proper axonal guidance and synaptogenesis without the isoform diversity found in insects. Differences in DSCAM function among several vertebrate species complicate the understanding of an evolutionarily conserved role during embryogenesis. We take advantage of the frog developmental model Xenopus tropicalis to study DSCAM function in early development by expression analysis and morpholino‐mediated knockdown. Our results indicate that DSCAM is expressed early in development and restricted to the head and nervous system. Knockdown of protein expression results in early morphogenetic phenotypes characterized by failed gastrulation and improper posterior neural tube closure. Our results reveal a specific, fundamental role of DSCAM in early morphogenetic movements, presumably through its well‐known role in homophilic cell adhesion. genesis 52:849–857, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

Regulation of multiple cell cycle events by Cdc14 homologues in vertebrates

Whereas early cytokinesis events have been relatively well studied, little is known about its final stage, abscission. The Cdc14 phosphatase is involved in the regulation of multiple cell cycle events, and in all systems studied Cdc14 misexpression leads to cytokinesis defects. In this work, we have cloned two CDC14 cDNA from Xenopus, including a previously unreported CDC14B homologue. We use Xenopus and human cell lines and demonstrate that localization of Cdc14 proteins is independent of both cell-type and species specificity. Ectopically expressed XCdc14A is centrosomal in interphase and localizes to the midbody in cytokinesis. By using XCdc14A misregulation, we confirm its control over different cell cycle events and unravel new functions during abscission. XCdc14A regulates the G1/S and G2/M transitions. We show that Cdc25 is an in vitro substrate for XCdc14A and might be its target at the G2/M transition. Upregulated wild-type or phosphatase-dead XCdc14A arrest cells in a late stage of cytokinesis, connected by thin cytoplasmic bridges. It does not interfere with central spindle formation, nor with the relocalization of passenger protein and centralspindlin complexes to the midbody. We demonstrate that XCdc14A upregulation prevents targeting of exocyst and SNARE complexes to the midbody, both essential for abscission to occur.     

Cytokinesis: a logical GAP

Two complexes localize to the central spindle and regulate completion of cytokinesis: one, centralspindlin, contains a kinesin-like protein and a Rho-family GAP; the second contains Aurora B kinase. Aurora B kinase is known to regulate localization of centralspindlin and may regulate the activity of the RhoGAP component of centralspindlin.     

The RhoGEF Pebble is required for cell shape changes during cell migration triggered by the Drosophila FGF receptor Heartless

The FGF receptor Heartless (HTL) is required for mesodermal cell migration in the Drosophila gastrula. We show that mesoderm cells undergo different phases of specific cell shape changes during mesoderm migration. During the migratory phase, the cells adhere to the basal surface of the ectoderm and exhibit extensive protrusive activity. HTL is required for the protrusive activity of the mesoderm cells. Moreover, the early phenotype of htl mutants suggests that HTL is required for the adhesion of mesoderm cells to the ectoderm. In a genetic screen we identified pebble (pbl) as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. We show that the function of PBL in cell migration is independent of the function of PBL in cytokinesis. Although RHO1 acts as a substrate for PBL in cytokinesis, compromising RHO1 function in the mesoderm does not block cell migration. These data suggest that the function of PBL in cell migration might be mediated through a pathway distinct from RHO1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. We show that PBL is autonomously required in the mesoderm for cell migration. Like HTL, PBL is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of HTL is unable to rescue the early cellular defects in pbl mutants, suggesting that PBL is required for the ability of HTL to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF PBL in HTL-dependent mesodermal cell migration.     

The cell adhesion-associated protein Git2 regulates morphogenetic movements during zebrafish embryonic development

Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.     

Cell migration regulates the kinetics of cytokinesis

Cytokinesis is the final stage of cell division in which the daughter cells separate. Although a growing body of evidence suggests that cell migration-induced traction forces may be required to provide physical assistance for daughter cells to dissociate during abscission, the role of cell migration in cytokinesis has not been directly elucidated. Recently, we have demonstrated that Crk and paxillin, which are pivotal components of the cell migration machinery, localize to the midbody and are essential for the abscission. These findings provided an important link between the cell migration and cytokinesis machineries and prompted us to dissect the role of cell migration in cytokinesis. We show that cell migration controls the kinetics of cleavage furrowing, midbody extension and abscission and coordinates proper subcellular redistribution of Crk and syntaxin-2 to the midbody after ingression.Key words: cell migration, cytokinesis, midbody, abscission, cleavage furrow, Crk, paxillin, syntaxin-2, ExoT     

Polo Kinase Interacts with RacGAP50C and Is Required to Localize the Cytokinesis Initiation Complex

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.     

The chromosomal passenger complex and centralspindlin independently contribute to contractile ring assembly

The chromosomal passenger complex (CPC) and centralspindlin are conserved cytokinesis regulators that localize to the spindle midzone, which forms between the separating chromosomes. Previous work placed the CPC and centralspindlin in a linear pathway that governs midzone formation. Using Caenorhabditis elegans embryos, we test whether there is a similar linear relationship between centralspindlin and the CPC in contractile ring constriction during cytokinesis. We show that simultaneous inhibition of the CPC kinase Aurora B(AIR-2) and the centralspindlin component MKLP1(ZEN-4) causes an additive constriction defect. Consistent with distinct roles for the proteins, inhibition of filamentous septin guanosine triphosphatases alleviates constriction defects in Aurora B(AIR-2)-inhibited embryos, whereas inhibition of Rac does so in MKLP1(ZEN-4)-inhibited embryos. Centralspindlin and the CPC are not required to enrich ring proteins at the cell equator but instead regulate formation of a compact mature ring. Therefore, in contrast to the linear midzone assembly pathway, centralspindlin and the CPC make independent contributions to control transformation of the sheet-like equatorial band into a ribbon-like contractile ring at the furrow tip.     

A remarkable new target gene for the dioxin receptor

The dioxin receptor (AhR) is possibly the best characterized xenobiotic receptor because of its essential role in mediating the harmful effects of highly toxic environmental pollutants. Despite the fact that AhR-dependent toxicity is a major environmental concern, compelling evidence is been lately produced unveiling novel and remarkable endogenous functions of AhR in cell physiology and tissue homeostasis. Adding to its role in cell proliferation and differentiation, AhR is also involved in the control of cell adhesion and migration, both highly relevant tasks in development and in disease states such as cancer. Interestingly, the effect of AhR on cell migration is cell-type specific because it can sustain or slow down cell motility. Here, I will comment on our recent report showing that AhR is a positive regulator of fibroblast cells migration. Besides characterizing the phenotype of such mesenchymal cells, the most important single finding of our study is that AhR uses the cytoskeleton regulator and oncogen Vav3 to signal through small Rho GTPases, ultimately leading to the physiological control of cell adhesion and migration. These data reveal that AhR activity is required to maintain signaling pathways governing normal cell function and open the question of whether AhR plays a role in cell migration during development and in pathological conditions such as tumor metastasis.     

Cell polarization and cytokinesis in budding yeast

Asymmetric cell division, which includes cell polarization and cytokinesis, is essential for generating cell diversity during development. The budding yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, and has thus served as an attractive model for unraveling the general principles of eukaryotic cell polarization and cytokinesis. Polarity development requires G-protein signaling, cytoskeletal polarization, and exocytosis, whereas cytokinesis requires concerted actions of a contractile actomyosin ring and targeted membrane deposition. In this chapter, we discuss the mechanics and spatial control of polarity development and cytokinesis, emphasizing the key concepts, mechanisms, and emerging questions in the field.     

A high-resolution shape fitting and simulation demonstrated equatorial cell surface softening during cytokinesis and its promotive role in cytokinesis

Different models for animal cell cytokinesis posit that the stiffness of the equatorial cortex is either increased or decreased relative to the stiffness of the polar cortex. A recent work has suggested that the critical cytokinesis signaling complex centralspindlin may reduce the stiffness of the equatorial cortex by inactivating the small GTPase Rac. To determine if such a reduction occurs and if it depends on centralspindlin, we devised a method to estimate cortical bending stiffness with high spatio-temporal resolution from in vivo cell shapes. Using the early Caenorhabditis elegans embryo as a model, we show that the stiffness of the equatorial cell surface is reduced during cytokinesis, whereas the stiffness of the polar cell surface remains stiff. The equatorial reduction of stiffness was compromised in cells with a mutation in the gene encoding the ZEN-4/kinesin-6 subunit of centralspindlin. Theoretical modeling showed that the absence of the equatorial reduction of stiffness could explain the arrest of furrow ingression in the mutant. By contrast, the equatorial reduction of stiffness was sufficient to generate a cleavage furrow even without the constriction force of the contractile ring. In this regime, the contractile ring had a supportive contribution to furrow ingression. We conclude that stiffness is reduced around the equator in a centralspindlin-dependent manner. In addition, computational modeling suggests that proper regulation of stiffness could be sufficient for cleavage furrow ingression.     

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confined TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of TbCentrin2 at the bilobe. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.     

GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. Here, we characterize the Drosophila melanogaster homologue of human GOLPH3 during cell division. We show that GOLPH3 accumulates at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. We demonstrate that the molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, we show that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results we propose that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, our studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis.     

Effect of mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> on proliferation and differentiation of wing cells

Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.     

LATITUDINAL VARIATION OF WING:THORAX SIZE RATIO AND WING-ASPECT RATIO IN DROSOPHILA MELANOGASTER

In dipterans, the wing-beat frequency, and, hence, the lift generated, increases linearly with ambient temperature. If flight performance is an important target of natural selection, higher wing:thorax size ratio and wing-aspect ratio should be favored at low temperatures because they increase the lift for a given body weight. We investigated this hypothesis by examining wing: thorax size ratio and wing-aspect ratio in Drosophila melanogaster collected from wild populations along a latitudinal gradient and in their descendants reared under standard laboratory conditions. In a subset of lines, we also studied the phenotypic plasticity of these traits in response to temperature. To examine whether the latitudinal trends in wing:thorax size ratio and wing-aspect ratio could have resulted from a correlated response to latitudinal selection on wing area, we investigated the correlated responses of these characters in lines artificially selected for wing area. In both the geographic and the artificially selected lines, wing:thorax size ratio and wing-aspect ratio decreased in response to increasing temperature during development. Phenotypic plasticity for either trait did not vary among latitudinal lines or selective regimes. Wing:thorax size ratio and wing-aspect ratio increased significantly with latitude in field-collected flies. The cline in wing:thorax size ratio had a genetic component, but the cline in wing-aspect ratio did not. Artificial selection for increased wing area led to a statistically insignificant correlated increase in wing:thorax size ratio and a decrease in wing-aspect ratio. Our observations are consistent with the hypotheses that high wing-thorax size ratio and wing aspect ratio are per se selectively advantageous at low temperatures.