CGEF-1 and CHIN-1 Regulate CDC-42 Activity during Asymmetric Division in the Caenorhabditis elegans Embryo

The anterior–posterior axis of the Caenorhabditis elegans embryo is elaborated at the one-cell stage by the polarization of the partitioning (PAR) proteins at the cell cortex. Polarization is established under the control of the Rho GTPase RHO-1 and is maintained by the Rho GTPase CDC-42. To understand more clearly the role of the Rho family GTPases in polarization and division of the early embryo, we constructed a fluorescent biosensor to determine the localization of CDC-42 activity in the living embryo. A genetic screen using this biosensor identified one positive (putative guanine nucleotide exchange factor [GEF]) and one negative (putative GTPase activating protein [GAP]) regulator of CDC-42 activity: CGEF-1 and CHIN-1. CGEF-1 was required for robust activation, whereas CHIN-1 restricted the spatial extent of CDC-42 activity. Genetic studies placed CHIN-1 in a novel regulatory loop, parallel to loop described previously, that maintains cortical PAR polarity. We found that polarized distributions of the nonmuscle myosin NMY-2 at the cell cortex are independently produced by the actions of RHO-1, and its effector kinase LET-502, during establishment phase and CDC-42, and its effector kinase MRCK-1, during maintenance phase. CHIN-1 restricted NMY-2 recruitment to the anterior during maintenance phase, consistent with its role in polarizing CDC-42 activity during this phase.

Download Full-text

Interaction between a Ras and a Rho GTPase Couples Selection of a Growth Site to the Development of Cell Polarity in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0426 ◽

2003 ◽

Vol 14 (12) ◽

pp. 4958-4970 ◽

Cited By ~ 67

Author(s):

Keith G. Kozminski ◽

Laure Beven ◽

Elizabeth Angerman ◽

Amy Hin Yan Tong ◽

Charles Boone ◽

...

Keyword(s):

Cell Polarity ◽

Rho Gtpase ◽

Cell Cortex ◽

Nucleotide Exchange ◽

Yeast Saccharomyces Cerevisiae ◽

Growth Site ◽

Ras Family ◽

Rho Family ◽

Polarity Establishment ◽

Selection Of

Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.

Download Full-text

ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration

The Journal of Cell Biology ◽

10.1083/jcb.200709033 ◽

2008 ◽

Vol 181 (2) ◽

pp. 351-365 ◽

Cited By ~ 87

Author(s):

Junji Yamauchi ◽

Yuki Miyamoto ◽

Jonah R. Chan ◽

Akito Tanoue

Keyword(s):

Cell Migration ◽

Schwann Cell ◽

Rho Gtpase ◽

Morphological Changes ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Schwann Cell Migration ◽

Subsequent Activation

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118–to–Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

Download Full-text

Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

The Journal of Cell Biology ◽

10.1083/jcb.153.6.1209 ◽

2001 ◽

Vol 153 (6) ◽

pp. 1209-1226 ◽

Cited By ~ 300

Author(s):

Karen Oegema ◽

Arshad Desai ◽

Sonja Rybina ◽

Matthew Kirkham ◽

Anthony A. Hyman

Keyword(s):

Caenorhabditis Elegans ◽

Chromosome Segregation ◽

Mitotic Chromosome ◽

Mitotic Chromosomes ◽

Cell Stage ◽

Kinetochore Assembly ◽

Chromosomal Passenger ◽

Complete Failure ◽

Spindle Microtubules ◽

The One

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

Download Full-text

Structure of the C-terminal guanine nucleotide exchange factor module of Trio in an autoinhibited conformation reveals its oncogenic potential

Science Signaling ◽

10.1126/scisignal.aav2449 ◽

2019 ◽

Vol 12 (569) ◽

pp. eaav2449 ◽

Cited By ~ 7

Author(s):

Sumit J. Bandekar ◽

Nadia Arang ◽

Ena S. Tully ◽

Brittany A. Tang ◽

Brenna L. Barton ◽

...

Keyword(s):

Rho Gtpase ◽

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Ph Domain ◽

Independent Manner ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Factor Module

The C-terminal guanine nucleotide exchange factor (GEF) module of Trio (TrioC) transfers signals from the Gαq/11subfamily of heterotrimeric G proteins to the small guanosine triphosphatase (GTPase) RhoA, enabling Gαq/11-coupled G protein–coupled receptors (GPCRs) to control downstream events, such as cell motility and gene transcription. This conserved signal transduction axis is crucial for tumor growth in uveal melanoma. Previous studies indicate that the GEF activity of the TrioC module is autoinhibited, with release of autoinhibition upon Gαq/11binding. Here, we determined the crystal structure of TrioC in its basal state and found that the pleckstrin homology (PH) domain interacts with the Dbl homology (DH) domain in a manner that occludes the Rho GTPase binding site, thereby suggesting the molecular basis of TrioC autoinhibition. Biochemical and biophysical assays revealed that disruption of the autoinhibited conformation destabilized and activated the TrioC module in vitro. Last, mutations in the DH-PH interface found in patients with cancer activated TrioC and, in the context of full-length Trio, led to increased abundance of guanosine triphosphate–bound RhoA (RhoA·GTP) in human cells. These mutations increase mitogenic signaling through the RhoA axis and, therefore, may represent cancer drivers operating in a Gαq/11-independent manner.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.200706167 ◽

2007 ◽

Vol 179 (6) ◽

pp. 1141-1148 ◽

Cited By ~ 51

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.

Download Full-text

Identification of a Novel Guanine Nucleotide Exchange Factor for the Rho GTPase

Journal of Biological Chemistry ◽

10.1074/jbc.271.41.25452 ◽

1996 ◽

Vol 271 (41) ◽

pp. 25452-25458 ◽

Cited By ~ 103

Author(s):

Matthew J. Hart ◽

Sanju Sharma ◽

Nadia elMasry ◽

Rong-Guo Qiu ◽

Peter McCabe ◽

...

Keyword(s):

Rho Gtpase ◽

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

Download Full-text

Uveal melanoma cells use ameboid and mesenchymal mechanisms of cell motility crossing the endothelium

Molecular Biology of the Cell ◽

10.1091/mbc.e20-04-0241 ◽

2021 ◽

pp. mbc.E20-04-0241

Author(s):

Michael D. Onken ◽

Kendall J. Blumer ◽

John A. Cooper

Keyword(s):

Immune Cells ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Direct Mechanism ◽

Guanine Nucleotide Exchange ◽

Amoeboid Motility ◽

Nucleotide Exchange Factor ◽

Oncogenic Mutations ◽

The One ◽

Pharmacologic Inhibition

Uveal melanomas (UM) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells is necessary for formation of metastases, and transendothelial migration (TEM) is a key step in extravasation. UM cells execute TEM via a stepwise process involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations that activate Gαq/11, and this activates TRIO, a guanine nucleotide exchange factor (GEF) for RhoA and Rac1. We found that pharmacologic inhibition of Gαq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 complex allowed cells to transmigrate without intercalation, a direct mechanism similar to the one often displayed by immune cells. BAP1-deficient (+/-) UM subclones displayed motility behaviors and increased levels of TEM, similar to the effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gαq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Download Full-text

A role for Gic1 and Gic2 in Cdc42 polarization

10.1101/407569 ◽

2018 ◽

Author(s):

Christine N. Daniels ◽

Trevin R. Zyla ◽

Daniel J. Lew

Keyword(s):

Feedback Loop ◽

Cell Types ◽

Effector Proteins ◽

Yeast Cells ◽

Cell Cortex ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Rho Family ◽

Cytoskeletal Elements

AbstractThe conserved Rho-family GTPase Cdc42 is a master regulator of polarity establishment in many cell types. Cdc42 becomes activated and concentrated in a region of the cell cortex, and recruits a variety of effector proteins to that site. In turn, many effectors participate in regulation of cytoskeletal elements in order to remodel the cytoskeleton in a polarized manner. The budding yeast Saccharomyces cerevisiae has served as a tractable model system for studies of cell polarity. In yeast cells, Cdc42 polarization involves a positive feedback loop in which effectors called p21-activated kinases (PAKs) act to recruit a Cdc42-directed guanine nucleotide exchange factor (GEF), generating more GTP-Cdc42 in areas that already have GTP-Cdc42. The GTPase-interacting components (GICs) Gic1 and Gic2 are also Cdc42 effectors, and have been implicated in regulation of the actin and septin cytoskeleton. However, we report that cells lacking GICs are primarily defective in polarizing Cdc42 itself, suggesting that they act upstream as well as downstream of Cdc42 in yeast. Our findings suggest that feedback pathways involving GTPase effectors may be more prevalent than had been appreciated.

Download Full-text

Rho GTPase–independent regulation of mitotic progression by the RhoGEF Net1

Molecular Biology of the Cell ◽

10.1091/mbc.e13-01-0061 ◽

2013 ◽

Vol 24 (17) ◽

pp. 2655-2667 ◽

Cited By ~ 9

Author(s):

Sarita Menon ◽

Wonkyung Oh ◽

Heather S. Carr ◽

Jeffrey A. Frost

Keyword(s):

Molecular Mechanisms ◽

Spindle Assembly Checkpoint ◽

Rho Gtpase ◽

Spindle Assembly ◽

Nucleotide Exchange ◽

Altered Expression ◽

Human Cancers ◽

Nucleotide Exchange Factor ◽

Chromosome Congression ◽

P21 Activated Kinase

Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily–specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

Download Full-text