Rho and Anillin-dependent Control of mDia2 Localization and Function in Cytokinesis

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

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mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e07-10-1086 ◽

2008 ◽

Vol 19 (5) ◽

pp. 2328-2338 ◽

Cited By ~ 124

Author(s):

Sadanori Watanabe ◽

Yoshikazu Ando ◽

Shingo Yasuda ◽

Hiroshi Hosoya ◽

Naoki Watanabe ◽

...

Keyword(s):

Rna Interference ◽

Mammalian Cell ◽

Equatorial Region ◽

Cleavage Furrow ◽

3T3 Cells ◽

Contractile Ring ◽

Binucleate Cells ◽

Dividing Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

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Inhibition of Cyclin-dependent Kinase 1 Induces Cytokinesis without Chromosome Segregation in an ECT2 and MgcRacGAP-dependent Manner

Journal of Biological Chemistry ◽

10.1074/jbc.m508007200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36502-36509 ◽

Cited By ~ 58

Author(s):

Fumihiko Niiya ◽

Xiaozhen Xie ◽

Kyung S. Lee ◽

Hiroki Inoue ◽

Toru Miki

Keyword(s):

Rna Interference ◽

Mammalian Cells ◽

Specific Inhibitor ◽

Small Gtpase ◽

Dominant Negative ◽

Cleavage Furrow ◽

Dependent Manner ◽

Cyclin Dependent Kinase ◽

Mitotic Spindles ◽

Initiation Mechanism

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.

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Interplay between MITF, PIAS3, and STAT3 in Mast Cells and Melanocytes

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10584-10592.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10584-10592 ◽

Cited By ~ 41

Author(s):

Amir Sonnenblick ◽

Carmit Levy ◽

Ehud Razin

Keyword(s):

Mast Cells ◽

Mrna Levels ◽

3T3 Cells ◽

Regulate Gene Expression ◽

Gp130 Receptor ◽

Microphthalmia Transcription Factor ◽

And Function ◽

Nih 3T3 ◽

Regulation Of Growth

ABSTRACT Microphthalmia transcription factor (MITF) and STAT3 are two transcription factors that play a major role in the regulation of growth and function in mast cells and melanocytes. In the present study, we explored the MITF-PIAS3-STAT3 network of interactions, how these interactions regulate gene expression, and how cytokine-mediated phosphorylation of MITF and STAT3 is involved in the in vivo interplay between these three proteins. In NIH 3T3 cells stimulated via gp130 receptor, transfected MITF was found to be phosphorylated at S409. Such phosphorylation of MITF leads to PIAS3 dissociation from MITF and its association with STAT3. Activation of mouse melanoma and mast cells through gp130 or c-Kit receptors induced the mobilization of PIAS3 from MITF to STAT3. In mast cells derived from MITF di/di mice, whose MITF lacks the Zip domain (PIAS3-binding domain), we found downregulation in mRNA levels of genes regulated by either MITF or STAT3. This regulatory mechanism is of considerable importance since it is likely to advance the deciphering of a role for MITF and STAT3 in mast cells and melanocytes.

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Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis

The Journal of Cell Biology ◽

10.1083/jcb.200408085 ◽

2005 ◽

Vol 168 (2) ◽

pp. 221-232 ◽

Cited By ~ 75

Author(s):

Fabian Oceguera-Yanez ◽

Kazuhiro Kimura ◽

Shingo Yasuda ◽

Chiharu Higashida ◽

Toshio Kitamura ◽

...

Keyword(s):

Rna Interference ◽

Rho Gtpase ◽

Dominant Negative ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Microtubule Attachment ◽

Nucleotide Exchange Factor ◽

Spindle Microtubules ◽

And Function

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.

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The adhesion-GPCR BAI1 shapes dendritic arbors via Bcr-mediated RhoA activation causing late growth arrest

eLife ◽

10.7554/elife.47566 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

Joseph G Duman ◽

Shalaka Mulherkar ◽

Yen-Kuei Tu ◽

Kelly C Erikson ◽

Christopher P Tzeng ◽

...

Keyword(s):

Hippocampal Neurons ◽

Rho Gtpase ◽

Regulatory Protein ◽

Growth Arrest ◽

Dendritic Morphology ◽

Small Gtpase ◽

Rhoa Activation ◽

Dependent Growth ◽

And Function ◽

Adhesion Gpcr

Dendritic arbor architecture profoundly impacts neuronal connectivity and function, and aberrant dendritic morphology characterizes neuropsychiatric disorders. Here, we identify the adhesion-GPCR BAI1 as an important regulator of dendritic arborization. BAI1 loss from mouse or rat hippocampal neurons causes dendritic hypertrophy, whereas BAI1 overexpression precipitates dendrite retraction. These defects specifically manifest as dendrites transition from growth to stability. BAI1-mediated growth arrest is independent of its Rac1-dependent synaptogenic function. Instead, BAI1 couples to the small GTPase RhoA, driving late RhoA activation in dendrites coincident with growth arrest. BAI1 loss lowers RhoA activation and uncouples it from dendrite dynamics, causing overgrowth. None of BAI1’s known downstream effectors mediates BAI1-dependent growth arrest. Rather, BAI1 associates with the Rho-GTPase regulatory protein Bcr late in development and stimulates its cryptic RhoA-GEF activity, which functions together with its Rac1-GAP activity to terminate arborization. Our results reveal a late-acting signaling pathway mediating a key transition in dendrite development.

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RNA Interference Vector Construction, Genetic Transformation and Function Analysis of Rice Transcription FactorOsMYB14

Chinese Journal of Appplied Environmental Biology ◽

10.3724/sp.j.1145.2013.00960 ◽

2013 ◽

Vol 19 (6) ◽

pp. 960

Author(s):

Yanwen MIAO ◽

Rong LI ◽

Fangjie XIONG ◽

Zhengming ZENG ◽

Xiangli NIU ◽

...

Keyword(s):

Rna Interference ◽

Genetic Transformation ◽

Function Analysis ◽

Vector Construction ◽

And Function

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TBC proteins: GAPs for mammalian small GTPase Rab?

Bioscience Reports ◽

10.1042/bsr20100112 ◽

2011 ◽

Vol 31 (3) ◽

pp. 159-168 ◽

Cited By ~ 115

Author(s):

Mitsunori Fukuda

Keyword(s):

Insulin Resistance ◽

Small Gtpases ◽

Structure And Function ◽

Small Gtpase ◽

Cell Cycle Regulators ◽

Gtpase Activating Protein ◽

Domain Family ◽

Protein Motif ◽

Conserved Domain ◽

And Function

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (TBC proteins) in other species are also expected to function as a certain Rab-GAP. More than 40 different TBC proteins are present in humans and mice, and recent accumulating evidence has indicated that certain mammalian TBC proteins actually function as a specific Rab-GAP. Some mammalian TBC proteins {e.g. TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1] and TBC1D4/AS160 (Akt substrate of 160 kDa)} play an important role in homoeostasis in mammals, and defects in them are directly associated with mouse and human diseases (e.g. leanness in mice and insulin resistance in humans). The present study reviews the structure and function of mammalian TBC proteins, especially in relation to Rab small GTPases.

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