Myosin II-interacting guanine nucleotide exchange factor promotes bleb retraction via stimulating cortex reassembly at the bleb membrane.

Blebs are involved in various biological processes such as cell migration, cytokinesis, and apoptosis. While the expansion of blebs is largely an intracellular pressure-driven process, the retraction of blebs is believed to be driven by RhoA activation that leads to the reassembly of the actomyosin cortex at the bleb membrane. However, it is still poorly understood how RhoA is activated at the bleb membrane. Here, we provide evidence demonstrating that myosin II-interacting guanine nucleotide exchange factor (MYOGEF) is implicated in bleb retraction via stimulating RhoA activation and the reassembly of an actomyosin network at the bleb membrane during bleb retraction. Interaction of MYOGEF with ezrin, a well-known regulator of bleb retraction, is required for MYOGEF localization to retracting blebs. Notably, knockout of MYOGEF or ezrin not only disrupts RhoA activation at the bleb membrane, but also interferes with nonmuscle myosin II localization and activation, as well as actin polymerization in retracting blebs. Importantly, MYOGEF knockout slows down bleb retraction. We propose that ezrin interacts with MYOGEF and recruits it to retracting blebs, where MYOGEF activates RhoA and promotes the reassembly of the cortical actomyosin network at the bleb membrane, thus contributing to the regulation of bleb retraction.

Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2.

Pitx2 is a bicoid-related homeobox transcription factor implicated in regulating left-right patterning and organogenesis. However, only a limited number of Pitx2 downstream target genes have been identified and characterized. Here we demonstrate that Pitx2 is a transcriptional repressor of DEP domain containing 1B (DEPDC1B). The first intron of the human and mouse DEP domain containing 1B genes contains multiple consensus DNA-binding sites for Pitx2. Chromatin immunoprecipitation assays revealed that Pitx2, along with histone deacetylase 1, was recruited to the first intron of Depdc1b. In contrast, RNAi-mediated depletion of Pitx2 not only enhanced the acetylation of histone H4 in the first intron of Depdc1b, but also increased the protein level of Depdc1b. Luciferase reporter assays also showed that Pitx2 could repress the transcriptional activity mediated by the first intron of human DEPDC1B. The GAP domain of DEPDC1B interacted with nucleotide-bound forms of RAC1 in vitro. In addition, exogenous expression of DEPDC1B suppressed RAC1 activation and interfered with actin polymerization induced by the guanine nucleotide exchange factor TRIO. Moreover, DEPDC1B interacted with various signaling molecules such as U2af2, Erh, and Salm. We propose that Pitx2-mediated repression of Depdc1b expression contributes to the regulation of multiple molecular pathways, such as Rho GTPase signaling.

Nonmuscle Myosin II Regulates the Morphogenesis of Metanephric Mesenchyme-Derived Immature Nephrons.

The kidney develops from reciprocal interactions between the metanephric mesenchyme and ureteric bud. The mesenchyme transforms into epithelia and forms complicated nephron structures, whereas the ureteric bud extends its pre-existing epithelial ducts. Although the roles are well established for extracellular stimuli, such as Wnt and Notch, it is unclear how the intracellular cytoskeleton regulates these morphogenetic processes. Myh9 and Myh10 encode nonmuscle myosin II heavy chains, and Myh9 mutations in humans are implicated in congenital kidney diseases and focal segmental glomerulosclerosis in adults. Here, we analyzed the roles of Myh9 and Myh10 in the developing kidney. Ureteric bud-specific depletion of Myh9 resulted in no apparent phenotypes, whereas mesenchyme-specific Myh9 deletion caused proximal tubule dilations and renal failure. Mesenchyme-specific Myh9/Myh10 mutant mice died shortly after birth and showed a severe defect in nephron formation. The nascent mutant nephrons failed to form a continuous lumen, which likely resulted from impaired apical constriction of the elongating tubules. In addition, nephron progenitors lacking Myh9/Myh10 or the possible interactor Kif26b were less condensed at midgestation and reduced at birth. Taken together, nonmuscle myosin II regulates the morphogenesis of immature nephrons derived from the metanephric mesenchyme and the maintenance of nephron progenitors. Our data also suggest that Myh9 deletion in mice results in failure to maintain renal tubules but not in glomerulosclerosis.

Intramolecular interactions between the Dbl homology (DH) domain and the carboxyl-terminal region of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) act as an autoinhibitory mechanism for the regulation of MyoGEF functions.

We have reported previously that nonmuscle myosin II-interacting guanine nucleotide exchange factor (MyoGEF) plays an important role in the regulation of cell migration and cytokinesis. Like many other guanine nucleotide exchange factors (GEFs), MyoGEF contains a Dbl homology (DH) domain and a pleckstrin homology domain. In this study, we provide evidence demonstrating that intramolecular interactions between the DH domain (residues 162-351) and the carboxyl-terminal region (501-790) of MyoGEF can inhibit MyoGEF functions. In vitro and in vivo pulldown assays showed that the carboxyl-terminal region (residues 501-790) of MyoGEF could interact with the DH domain but not with the pleckstrin homology domain. Expression of a MyoGEF carboxyl-terminal fragment (residues 501-790) decreased RhoA activation and suppressed actin filament formation in MDA-MB-231 breast cancer cells. Additionally, Matrigel invasion assays showed that exogenous expression of the MyoGEF carboxyl-terminal region decreased the invasion activity of MDA-MB-231 cells. Moreover, coimmunoprecipitation assays showed that phosphorylation of the MyoGEF carboxyl-terminal region by aurora B kinase interfered with the intramolecular interactions of MyoGEF. Furthermore, expression of the MyoGEF carboxyl-terminal region interfered with RhoA localization during cytokinesis and led to an increase in multinucleation. Together, our findings suggest that binding of the carboxyl-terminal region of MyoGEF to its DH domain acts as an autoinhibitory mechanism for the regulation of MyoGEF activation.

Phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) at threonine 544 by aurora B kinase promotes the binding of polo-like kinase 1 to MyoGEF.

We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.

Non-muscle myosin IIB is essential for cytokinesis during male meiotic cell divisions.

Cytokinesis, the final stage of cell division, bisects the cytoplasm into two daughter cells. In mitotic cells, this process depends on the activity of non-muscle myosin II (NMII), a family of actin-binding motor-proteins that participate in the formation of the cleavage furrow. The relevance of NMII for meiotic cell division, however, is poorly understood. The NMII family consists of three members, NMIIA, NMIIB, and NMIIC, containing different myosin heavy chains (MYH9, MYH10, and MYH14, respectively). We find that a single non-muscle myosin II, NMIIB, is required for meiotic cytokinesis in male but not female mice. Specifically, NMIIB-deficient spermatocytes exhibit cytokinetic failure in meiosis I, resulting in bi-nucleated secondary spermatocytes. Additionally, cytokinetic failure at meiosis II gives rise to bi-nucleated or even tetra-nucleated spermatids. These multi-nucleated spermatids fail to undergo normal differentiation, leading to male infertility. In spite of the presence of multiple non-muscle myosin II isoforms, we demonstrate that a single member, NMIIB, plays an essential and non-redundant role in cytokinesis during meiotic cell divisions of the male germline.

Role of a novel coiled-coil domain-containing protein CCDC69 in regulating central spindle assembly.

The formation of the central spindle (or the spindle midzone) is essential for cytokinesis in animal cells. In this study, we report that coiled-coil domain-containing protein 69 (CCDC69) is implicated in controlling the assembly of central spindles and the recruitment of midzone components. Exogenous expression of CCDC69 in HeLa cells interfered with microtubule polymerization and disrupted the formation of bipolar mitotic spindles. Endogenous CCDC69 proteins were localized to the central spindle during anaphase. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and disrupted the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and polo-like kinase 1 (Plk1) at the central spindle. Aurora B kinase was found to bind to CCDC69 and this binding depended on the coiled-coil domains at the C-terminus of CCDC69. Further, disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Our results indicate that CCDC69 acts as a scaffold to regulate the recruitment of midzone components and the assembly of central spindles.

GIPC1 interacts with MyoGEF and promotes MDA-MB-231 breast cancer cell invasion.

GIPC1/synectin, a single PDZ domain-containing protein, binds to numerous proteins and is involved in multiple biological processes, including cell migration. We reported previously that MyoGEF, a guanine nucleotide exchange factor, plays a role in regulating breast cancer cell polarization and invasion. Here, we identify GIPC1 as an interacting partner of MyoGEF. Both in vitro and in vivo binding assays show that the GIPC1 PDZ domain binds to the PDZ-binding motif at the C terminus of MyoGEF. Immunofluorescence analysis shows that GIPC1 and MyoGEF colocalize to the cell leading edge. Depletion of GIPC1 by RNAi in MDA-MB-231 cells causes cells to shift from a polarized to a rounded morphology. Matrigel invasion assays show that RNAi-mediated depletion of GIPC1 dramatically decreases MDA-MB-231 cell invasion. Notably, an anti-MyoGEF peptide antibody, whose epitope is located at the C terminus of MyoGEF, interferes with GIPC1-MyoGEF complex formation. Treatment of MDA-MB-231 cells with the anti-MyoGEF peptide antibody disrupts cell polarization and invasion. Thus, our results suggest that GIPC1-MyoGEF complex formation plays an important role in regulating MDA-MB-231 breast cancer cell polarization and invasion.

Conditional ablation of nonmuscle myosin II-B delineates heart defects in adult mice.

RATIONALE: Germline ablation of the cytoskeletal protein nonmuscle myosin II (NMII)-B results in embryonic lethality, with defects in both the brain and heart. Tissue-specific ablation of NMII-B by a Cre recombinase strategy should prevent embryonic lethality and permit study of the function of NMII-B in adult hearts. OBJECTIVE: We sought to understand the function of NMII-B in adult mouse hearts and to see whether the brain defects found in germline-ablated mice influence cardiac development. METHODS AND RESULTS: We used a loxP/Cre recombinase strategy to specifically ablate NMII-B in the brains or hearts of mice. Mice ablated for NMII-B in neural tissues die between postnatal day 12 and 22 without showing cardiac defects. Mice deficient in NMII-B only in cardiac myocytes (B(alphaMHC)/B(alphaMHC) mice) do not show brain defects. However, B(alphaMHC)/B(alphaMHC) mice display novel cardiac defects not seen in NMII-B germline-ablated mice. Most of the B(alphaMHC)/B(alphaMHC) mice are born with enlarged cardiac myocytes, some of which are multinucleated, reflecting a defect in cytokinesis. Between 6 to 10 months, they develop a cardiomyopathy that includes interstitial fibrosis and infiltration of the myocardium and pericardium with inflammatory cells. Four of 5 B(alphaMHC)/B(alphaMHC) hearts develop marked widening of intercalated discs. CONCLUSIONS: By avoiding the embryonic lethality found in germline-ablated mice, we were able to study the function of NMII-B in adult mice and show that absence of NMII-B in cardiac myocytes results in cardiomyopathy in the adult heart. We also define a role for NMII-B in maintaining the integrity of intercalated discs.

Centrosome/spindle pole-associated protein regulates cytokinesis via promoting the recruitment of MyoGEF to the central spindle.

Cooperative communications between the central spindle and the contractile ring are critical for the spatial and temporal regulation of cytokinesis. Here we report that MyoGEF, a guanine nucleotide exchange factor that localizes to the central spindle and cleavage furrow, interacts with centrosome/spindle pole-associated protein (CSPP), which is concentrated at the spindle pole and central spindle during mitosis and cytokinesis. Both in vitro and in vivo pulldown assays show that MyoGEF interacts with CSPP. The C-terminus of MyoGEF and N-terminus of CSPP are required for their interaction. Immunofluorescence analysis indicates that MyoGEF and CSPP colocalize at the central spindle. Depletion of CSPP or MyoGEF by RNA-interference (RNAi) not only causes defects in mitosis and cytokinesis, such as metaphase arrest and furrow regression, but also mislocalization of nonmuscle myosin II with a phosphorylated myosin regulatory light chain (p-MRLC). Importantly, CSPP depletion by RNAi interferes with MyoGEF localization at the central spindle. Finally, MyoGEF interacts with ECT2, and RNAi-mediated depletion of MyoGEF leads to mislocalization of ECT2 and RhoA during cytokinesis. Therefore, we propose that CSPP interacts with and recruits MyoGEF to the central spindle, where MyoGEF contributes to the spatiotemporal regulation of cytokinesis.

Phosphorylation of MyoGEF on Thr-574 by Plk1 promotes MyoGEF localization to the central spindle.

We reported previously that a guanine nucleotide exchange factor, MyoGEF, localizes to the central spindle, activates RhoA, and is required for cytokinesis. In this study, we have found that Plk1 (polo-like kinase 1) can phosphorylate MyoGEF, thereby recruiting MyoGEF to the central spindle as well as enhancing MyoGEF activity toward RhoA. The in vitro kinase assay shows that Plk1 can phosphorylate MyoGEF on threonine 574. Immunoprecipitation/immunoblot analysis demonstrates that mutation of threonine 574 to alanine dramatically decreases threonine phosphorylation of MyoGEF in transfected HeLa cells, suggesting that threonine 574 is phosphorylated in vivo. Consistent with these observations, immunofluorescence shows that Plk1 and MyoGEF colocalize at the spindle pole and central spindle during mitosis and cytokinesis. Importantly, RNA interference-mediated depletion of Plk1 interferes with the localization of MyoGEF at the spindle pole and central spindle. Moreover, mutation of threonine 574 to alanine in MyoGEF or depletion of Plk1 by RNA interference leads to a decrease in MyoGEF activity toward RhoA in HeLa cells. Therefore, our results suggest that Plk1 can regulate MyoGEF activity and localization, contributing to the regulation of cytokinesis.

A novel guanine nucleotide exchange factor MyoGEF is required for cytokinesis.

The cleavage furrow is created by an actomyosin contractile ring that is regulated by small GTPase proteins such as Rac1 and RhoA. Guanine nucleotide exchange factors (GEFs) are positive regulators of the small GTPase proteins and have been implicated as important factors in regulating cytokinesis. However, it is still unclear how GEFs regulate the contractile ring during cytokinesis in mammalian cells. Here we report that a novel GEF, which is termed MyoGEF (myosin-interacting GEF), interacts with non-muscle myosin II and exhibits activity toward RhoA. MyoGEF and non-muscle myosin II colocalize to the cleavage furrow in early anaphase cells. Disruption of MyoGEF expression in U2OS cells by RNA interference (RNAi) results in the formation of multinucleated cells. These results suggest that MyoGEF, RhoA, and non-muscle myosin II act as a functional unit at the cleavage furrow to advance furrow ingression during cytokinesis.

Pitx2a binds to human papillomavirus type 18 E6 protein and inhibits E6-mediated P53 degradation in HeLa cells.

Binding of high risk human papillomavirus (HPV) E6 protein to E6-associated protein (E6AP), a cellular ubiquitin-protein ligase, enables E6AP to ubiquitinate p53, leading to p53 degradation in cervical cancer cells such as HeLa cells. Here we report that Pitx2a, a bicoid-type homeodomain transcription factor, can bind to HPV E6 protein and inhibit E6/E6AP-mediated p53 degradation. Deletion of the Pitx2a homeodomain abrogates its ability to bind to HPV E6 protein and to induce p53 accumulation in HeLa cells, suggesting that the homeodomain of Pitx2a is essential for inhibition of E6/E6AP-mediated p53 degradation. Recombinant Pitx2a can also block E6/E6AP-mediated p53 degradation in vitro, indicating that this function of Pitx2a is independent of its transcription activity. Pitx2a does not regulate Hdm2-mediated p53 degradation, because Pitx2a does not affect p53 protein levels in HPV-negative cells, such as HCT116, U2OS, and C33A cells. In addition, Pitx2a-induced p53 is transcriptionally active and maintains its specific DNA binding activity in HeLa cells. Taken together, these findings suggest that, by binding to E6, Pitx2a interferes with E6/E6AP-mediated p53 degradation, leading to the accumulation of functional p53 protein in HeLa cells.

Basic mechanism of three-dimensional collagen fibre transport by fibroblasts.

Collagen remodelling by fibroblasts has a crucial role in organizing tissue structures that are essential to motility during wound repair, development and regulation of cell growth. However, the mechanism of collagen fibre movement in three-dimensional (3D) matrices is not understood. Here, we show that fibroblast lamellipodia extend along held collagen fibres, bind, and retract them in a 'hand-over-hand' cycle, involving alpha2beta1 integrin. Wild-type fibroblasts move collagen fibres three to four times farther per cycle than fibroblasts lacking myosin II-B (myosin II-B(-/-)). Similarly, myosin II-B(-/-) fibroblasts contract 3D collagen gels threefold less than controls. On two-dimensional (2D) substrates, however, rates of collagen bead and cell movement are not affected by loss of myosin II-B. Green fluorescent protein (GFP)-tagged myosin II-B, but not II-A, restores normal function in knockout cells and localizes to cell processes, whereas myosin II-A is more centrally located. Additionally, GFP-myosin II-B moves out to the periphery and back during hand-over-hand fibre movement, whereas on 2D collagen, myosin II-B is more centrally distributed. Thus, we suggest that cyclic myosin II-B assembly and contraction in lamellipodia power 3D fibre movements.

Pitx2a expression alters actin-myosin cytoskeleton and migration of HeLa cells through Rho GTPase signaling.

We ectopically expressed the transcription factor Pitx2a, one of the Pitx2 isoforms, in HeLa cells by using a tetracycline-inducible expression system and examined whether Pitx2a was capable of modulating Rho GTPase signaling and altering the cell's cytoskeleton. Ectopic expression of Pitx2a induced actin-myosin reorganization, leading to increased cell spreading, suppression of cell migration, and the strengthening of cell-cell adhesion, marked by the accumulation and localization of beta-catenin and N-cadherin to the sites of cell-cell contacts. Moreover, Pitx2a expression resulted in activation of the Rho GTPases Rac1 and RhoA, and the dominant negative Rac1 mutant N17Rac1 inhibited cell spreading and disrupted localization of beta-catenin to the sites of cell-cell contacts. Both reorganization of actin-myosin and cell spreading require phosphatidylinositol 3-kinase activity, which is also necessary for activation of the Rho GTPase proteins. Pitx2a induced the expression of Trio, a guanine nucleotide exchange factor for Rac1 and RhoA, which preceded cell spreading, and the expression of Trio protein was down-regulated after the changes in cell spreading and cell morphology were initiated. In addition, Pitx2a also induces cell cycle arrest at G0/G1, most likely due to the accumulation of the tumor suppressor proteins p53 and p21. Our data indicate that the transcriptional activities initiated in the nucleus by Pitx2a result in profound changes in HeLa cell morphology, migration, and proliferation.