scholarly journals Novel Functions of Ect2 in Polar Lamellipodia Formation and Polarity Maintenance during “Contractile Ring-Independent” Cytokinesis in Adherent Cells

2008 ◽  
Vol 19 (1) ◽  
pp. 8-16 ◽  
Author(s):  
Masamitsu Kanada ◽  
Akira Nagasaki ◽  
Taro Q.P. Uyeda
Keyword(s):  
Mammalian Cells ◽  
Polar Regions ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ht1080 Cells ◽  
Lamellipodia Formation

Some mammalian cells are able to divide via both the classic contractile ring-dependent method (cytokinesis A) and a contractile ring-independent, adhesion-dependent method (cytokinesis B). Cytokinesis A is triggered by RhoA, which, in HeLa cells, is activated by the guanine nucleotide-exchange factor Ect2 localized at the central spindle and equatorial cortex. Here, we show that in HT1080 cells undergoing cytokinesis A, Ect2 does not localize in the equatorial cortex, though RhoA accumulates there. Moreover, Ect2 depletion resulted in only modest multinucleation of HT1080 cells, enabling us to establish cell lines in which Ect2 was constitutively depleted. Thus, RhoA is activated via an Ect2-independent pathway during cytokinesis A in HT1080 cells. During cytokinesis B, Ect2-depleted cells showed narrower accumulation of RhoA at the equatorial cortex, accompanied by compromised pole-to-equator polarity, formation of ectopic lamellipodia in regions where RhoA normally would be distributed, and delayed formation of polar lamellipodia. Furthermore, C3 exoenzyme inhibited equatorial RhoA activation and polar lamellipodia formation. Conversely, expression of dominant active Ect2 in interphase HT1080 cells enhanced RhoA activity and suppressed lamellipodia formation. These results suggest that equatorial Ect2 locally suppresses lamellipodia formation via RhoA activation, which indirectly contributes to restricting lamellipodia formation to polar regions during cytokinesis B.

scholarly journals PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

2008 ◽  
Vol 180 (1) ◽  
pp. 187-203 ◽  
Author(s):  
Yangmi Lim ◽  
Ssang-Taek Lim ◽  
Alok Tomar ◽  
Margaret Gardel ◽  
Joie A. Bernard-Trifilo ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cellular Contact

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

scholarly journals A GAP that Divides

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1788 ◽  
Author(s):  
Angika Basant ◽  
Michael Glotzer
Keyword(s):  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
C Elegans ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Parallel Pathway ◽  
Mutant Phenotypes

Cytokinesis in metazoan cells is mediated by an actomyosin-based contractile ring that assembles in response to activation of the small GTPase RhoA. The guanine nucleotide exchange factor that activates RhoA during cytokinesis, ECT-2, is highly regulated. In most metazoan cells, with the notable exception of the early Caenorhabditis elegans embryo, RhoA activation and furrow ingression require the centralspindlin complex. This exception is due to the existence of a parallel pathway for RhoA activation in C. elegans. Centralspindlin contains CYK-4 which contains a predicted Rho family GTPase-activating protein (GAP) domain. The function of this domain has been the subject of considerable debate. Some publications suggest that the GAP domain promotes RhoA activation (for example, Zhang and Glotzer, 2015; Loria, Longhini and Glotzer, 2012), whereas others suggest that it functions to inactivate the GTPase Rac1 (for example, Zhuravlev et al., 2017). Here, we review the mechanisms underlying RhoA activation during cytokinesis, primarily focusing on data in C. elegans. We highlight the importance of considering the parallel pathway for RhoA activation and detailed analyses of cyk-4 mutant phenotypes when evaluating the role of the GAP domain of CYK-4.

scholarly journals PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells

2007 ◽  
Vol 179 (6) ◽  
pp. 1141-1148 ◽  
Author(s):  
Kit Wong ◽  
Alexandra Van Keymeulen ◽  
Henry R. Bourne
Keyword(s):  
Rho Gtpase ◽  
Myosin Ii ◽  
Nucleotide Exchange ◽  
Dependent Protein Kinase ◽  
Rhoa Activity ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Long Tails ◽  
Dependent Protein ◽  
Atpase Inhibition

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.

scholarly journals Cloning and expression of cDNAs for the β subunit of eukaryotic initiation factor-2B, the guanine nucleotide exchange factor for eukaryotic initiation factor-2

1995 ◽  
Vol 309 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
B L Craddock ◽  
N T Price ◽  
C G Proud
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mammalian Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

A key control point in the initiation of protein synthesis in mammalian cells is the recycling of eukaryotic initiation factor (eIF)-2 by the guanine nucleotide exchange factor eIF-2B. In mammalian cells, eIF-2B is a complex of five different subunits termed epsilon, delta, gamma, beta and alpha. To clone cDNAs for the beta subunit of rabbit eIF-2B, amino acid sequence data was first obtained and used to design redundant oligonucleotide primers for use in PCR. PCR products were used to screen a rabbit liver cDNA library in lambda gt11 to obtain full-length cDNAs for eIF-2B beta. The cDNAs were sequenced completely on both strands and revealed an open reading frame encoding a predicted 351-amino acid polypeptide of 39.0 kDa. The molecular mass and pI (5.99) of the predicted protein agree well with the properties of eIF-2B beta purified from rabbit reticulocytes. In vitro transcription/-translation of the cDNAs gave rise to a product that migrated at a position indistinguishable from that of this subunit of the purified protein. The amino acid sequence shows a high degree of similarity to that of GCD7, a Saccharomyces cerevisiae protein thought to be equivalent to mammalian eIF-2B beta. Northern-blot analysis revealed a single major mRNA species for eIF-2B beta in each of the four rabbit tissues tested.

scholarly journals mTrs130 Is a Component of a Mammalian TRAPPII Complex, a Rab1 GEF That Binds to COPI-coated Vesicles

2009 ◽  
Vol 20 (19) ◽  
pp. 4205-4215 ◽  
Author(s):  
Akinori Yamasaki ◽  
Shekar Menon ◽  
Sidney Yu ◽  
Jemima Barrowman ◽  
Timo Meerloo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Golgi Compartment ◽  
A Subunit ◽  
Protein Particle ◽  
First Time

The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to γ1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes.

scholarly journals GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication

2009 ◽  
Vol 83 (22) ◽  
pp. 11940-11949 ◽  
Author(s):  
Kjerstin H. W. Lanke ◽  
Hilde M. van der Schaar ◽  
George A. Belov ◽  
Qian Feng ◽  
Daniël Duijsings ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mdck Cells ◽  
Critical Role ◽  
Brefeldin A ◽  
Rna Replication ◽  
Coxsackievirus B3 ◽  
Viral Rna ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.

scholarly journals Vesicles and actin are targeted to the cleavage furrow via furrow microtubules and the central spindle

2008 ◽  
Vol 181 (5) ◽  
pp. 777-790 ◽  
Author(s):  
Roger Albertson ◽  
Jian Cao ◽  
Tao-shih Hsieh ◽  
William Sullivan
Keyword(s):  
Drosophila Melanogaster ◽  
Vesicle Transport ◽  
Cleavage Furrow ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Distinct Population ◽  
Contractile Ring ◽  
Central Spindle ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

During cytokinesis, cleavage furrow invagination requires an actomyosin-based contractile ring and addition of new membrane. Little is known about how this actin and membrane traffic to the cleavage furrow. We address this through live analysis of fluorescently tagged vesicles in postcellularized Drosophila melanogaster embryos. We find that during cytokinesis, F-actin and membrane are targeted as a unit to invaginating furrows through formation of F-actin–associated vesicles. F-actin puncta strongly colocalize with endosomal, but not Golgi-derived, vesicles. These vesicles are recruited to the cleavage furrow along the central spindle and a distinct population of microtubules (MTs) in contact with the leading furrow edge (furrow MTs). We find that Rho-specific guanine nucleotide exchange factor mutants, pebble (pbl), severely disrupt this F-actin–associated vesicle transport. These transport defects are a consequence of the pbl mutants' inability to properly form furrow MTs and the central spindle. Transport of F-actin–associated vesicles on furrow MTs and the central spindle is thus an important mechanism by which actin and membrane are delivered to the cleavage furrow.

scholarly journals Local RhoA Activation Induces Cytokinetic Furrows Independent of Spindle Position and Cell Cycle Stage

2016 ◽  
Author(s):  
Elizabeth Wagner ◽  
Michael Glotzer
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Contractile Ring ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Ring Assembly ◽  
Interphase Cells ◽  
A Site ◽  
Spindle Position ◽  
Rapid Activation

Cytokinetic cleavage furrows assemble during anaphase at a site that is dictated by the position of the spindle. The GTPase RhoA promotes contractile ring assembly and furrow ingression during cytokinesis. While many factors that regulate RhoA during cytokinesis have been characterized, the spatiotemporal regulatory logic remains undefined. It is not known whether a local zone of RhoA activity is sufficient to induce furrow formation or whether the spindle modulates furrow assembly through other pathways. Similarly, it is not known whether the entire cortex is responsive to RhoA, nor whether contractile ring assembly is cell cycle regulated. Here, we have developed an optogenetic probe to gain tight spatial and temporal control of RhoA activity in mammalian cells and demonstrate that cytokinetic furrowing is primarily regulated at the level of RhoA activation. Light-mediated recruitment of a RhoGEF domain to the plasma membrane leads to rapid activation of RhoA, leading to assembly of cytokinetic furrows that partially ingress. Furthermore, furrow formation in response to RhoA activation is not spatially or temporally restricted. RhoA activation is sufficient to generate furrows at both the cell equator and at cell poles, in both metaphase and anaphase. Remarkably, furrow formation can be initiated in rounded interphase cells, but not adherent cells. These results indicate RhoA activation is sufficient to induce assembly of functional contractile rings and that cell rounding facilitates furrow formation.

scholarly journals Myosin II–interacting guanine nucleotide exchange factor promotes bleb retraction via stimulating cortex reassembly at the bleb membrane

2018 ◽  
Vol 29 (5) ◽  
pp. 643-656 ◽  
Author(s):  
Meng Jiao ◽  
Di Wu ◽  
Qize Wei
Keyword(s):  
Actin Polymerization ◽  
Myosin Ii ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Nonmuscle Myosin Ii

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.

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