Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

Yang, Linda; Wang, Lei; Zheng, Yi

doi:10.1091/mbc.e06-05-0466

Cited by 138 publications

(146 citation statements)

References 34 publications

(45 reference statements)

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“…Note also that 3 and 5 pg of DN-Cdc42 mRNA alone dramatically impacted the morphology and decreased the survival of the embryos to 20-to 24-somite stages. These results suggest not only that Cdc42 is an essential downstream effecter of p120 catenin d1 for normal cell migration, but also that the ability of Cdc42 to initiate directional polarity of migrating cells by localized GDP to GTP exchange (Etteinne-Manneville, 2004, 2008Etteinne-Manneville and Hall 2001;Yang et al, 2006;Johnson et al, 2010) or to cycle between its active GTP-bound and inactive GDPbound states is an essential feature in this signaling pathway.…”

Section: Defects From Knockdown Of P120 Catenin D1 Persist In Later-smentioning

confidence: 89%

“…To investigate our hypothesis that the polarized regulation of Rho GTPases by p120 catenin d1 is necessary during gastrulation, we examined the ability of various forms of Cdc42 to rescue the d1 splice-MO phenotype. Cdc42 is a small (191 amino acids) member of the Rho GTPases that is crucial for the formation of filopodia along the leading edge of migrating vertebrate cells (Etienne-Manneville and Hall, 2001;Choi and Han, 2003;Etienne-Manneville, 2004, 2008Yang Developmental Dynamics Fig. 5.…”

Section: Defects From Knockdown Of P120 Catenin D1 Persist In Later-smentioning

confidence: 99%

“…Our observations that DN-RhoA can rescue the knockdown of p120 catenin d1 show that a release from adhesion also promotes somite cell migration at low concentrations of p120 catenin d1. Since Cdc42 GTPase initiates the polarity of cells (Etteinne-Manneville, 2004, 2008Etteinne-Manneville and Hall, 2001;Yang et al, 2006;Johnson et al, 2010), the inability of CA-Cdc42 GTPase to rescue the knockdown of p120 catenin d1 is not surprising. CACdc42 would compete with endogenous WT-Cdc42 and block the stimulation of actin polymerization by WTCdc42 GTPase.…”

Section: Developmental Dynamicsmentioning

confidence: 99%

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Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Hsu

Muerdter

Knickerbocker

et al. 2012

Developmental Dynamics

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Background: We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin-mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. Results: During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenind1 mRNAs with a splice-site morpholino caused lateral widening and anterior-posterior shortening of the presomitic mesoderm and somites and a shortened anterior-posterior axis. These phenotypes indicate a cell-migration effect. Co-injection of low amounts of wild-type Cdc42 or wild-type Rac1 or dominant-negative RhoA mRNAs, but not constitutively-active Cdc42 mRNA, rescued these p120 catenin d1-depleted embryos. Conclusions: These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development. Developmental Dynamics 241:1545-1561, 2012. V C 2012 Wiley Periodicals Inc.Key words: p120 Catenin (CTNND1); ARVCF; Delta-catenin (CTNND2b); Cdc42 GTPase; Rac1 GTPase, RhoA GTPase; gastrulation; presomitic mesoderm; somites; zebrafish Key findings p120 catetin is required for extension of the dorsal axis and normal migration of the presomitic mesoderm. Cdc42 and Rac1 GTPases are downstream of p120 catenin d1 signaling and require exchange of GTP for GDP. Local stimulation of the exchange of GTP for GDP in Cdc42 and Rac GTPases mediates directional migration of the presomitic mesoderm. A balance of the amount of p120 catenin d1 and localized activation or turnover of Cdc42, Rac1, and Rho GTPase are required for normal zebrafish cell migration. Accepted 31 July 2012 Developmental DynamicsABBREVIATIONS Ab antibody ARVCF armadillo repeat gene deleted in velo-cardio-facial syndrome CA constitutively active Chr chromosome C(t) relative amount of RT-PCR product hpf hours post-fertilization DN dominant negative d1 splice-MO antisense morpholino oligonucleotide to the 12 th splice site of zebrafish p120 catenin d1 p120 catenin d1 (CTNND1) also called p120 catenin, Xenopus p120 catenin is a CTNND1 p120 catenin d2b (CTNND2b) also called Delta-catenin Rok1 Rho kinase1 RT reverse transcriptase minus-RT controls without reverse transcriptase qRT-PCR quantitative real-time PCR WT wild-type Xp120 catenin mRNA Xenopus p120 catenin d1 mRNA.Additional Supporting Information may be found in the online version of this article.

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Section: Defects From Knockdown Of P120 Catenin D1 Persist In Later-smentioning

confidence: 89%

Section: Defects From Knockdown Of P120 Catenin D1 Persist In Later-smentioning

confidence: 99%

Section: Developmental Dynamicsmentioning

confidence: 99%

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Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Hsu

Muerdter

Knickerbocker

et al. 2012

Developmental Dynamics

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“…We therefore wondered whether cytokinesis represented an isoform-specific function of the ubiquitously expressed Dyn2. To test this possibility we generated retroviruses encoding HA-Dyn1 and HA-Dyn2 using a retroviral vector containing an internal ribosomal entry site (IRES) upstream of GFP according to the strategy of Yang et al (2006). We used FACS to select GFP-expressing cells that also expressed low, near endogenous levels of HA-dynamin (see below).…”

Section: Dyn2 Ko Cells Exhibit Growth and Cytokinesis Defectsmentioning

confidence: 99%

“…HA-tagged human Dyn1 (ba) or rat Dyn2 (aa, ab, ba, and bb) were cloned into the retrovirus vector pMIEG3 containing an internal ribosome entry site allowing expression of enhanced green fluorescent protein (MIEG3-EGFP; Yang et al, 2006). Alternatively, HA-Dyn1 (ba) or HA-Dyn2 (ba) were fused to GFP on their C-termini and used to generate retroviruses.…”

Section: Retroviral Transduction and Fluorescence-activated Cell Sortingmentioning

confidence: 99%

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

Liu

Surka

Schroeter

et al. 2008

MBoC

125

163

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Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform-and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGFstimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. INTRODUCTIONDynamin (Dyn) is an ϳ100-kDa multidomain GTPase that was first identified as a microtubule binding and bundling protein (Shpetner and Vallee, 1989). Subsequently, dynamin was found to be the mammalian homologue of the Drosophila protein shibire, mutations in which block endocytosis, including synaptic vesicle recycling (Chen et al., 1991;van der Bliek and Meyerowitz, 1991). Dynamin is conserved throughout higher eukaryotes. There is a single gene in Drosophila and Caenorhabditis elegans, but there are three dynamin isoforms in mammals: Dyn1, which is specifically expressed in neurons; Dyn2, which is ubiquitously expressed; and Dyn3, which is mainly expressed in the brain and testes (Urrutia et al., 1997, Ferguson et al., 2007.The best-studied cellular function of dynamin is its involvement in clathrin-mediated endocytosis (CME; Hinshaw, 2000;Sever et al., 2000;Praefcke and McMahon, 2004). However, dynamin has also been implicated in several other membrane-trafficking events including both caveolae-mediated and clathrin-and caveolin-independent endocytic pathways Oh et al., 1998;Lamaze et al., 2001;Pelkmans et al., 2002), phagocytosis (Gold et al., 1999;Yu et al., 2006), macropinocytosis (Schlunck et al., 2004), and trafficking from the trans-Golgi network (TGN; Jones et al., 1998;Kreitzer et al., 2000;Bonazzi et al., 2005). Because these functions have mostly emerged from studying the effects of overexpression of dominant-negative dynamin mutants, they may reflect indirect or nonspecific effects. Moreover, it is not known whether different dynamin isoforms and/or splice varia...

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Rho GTPase Function in Cell Morphogenesis

Pedersen

Brakebusch

2010

Encyclopedia of Life Sciences

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Cell morphogenesis is the development of a specific cell morphology, which includes the acquisition of cell shape, cell polarity, cell–cell and cell–ECM (extracellular matrix) contacts. Cell morphogenesis is dependent on the interaction of the cell with its environment and therefore not a cell autonomous process. Crucial for cell morphogenesis is the regulation of the actin cytoskeleton. Furthermore, regulation of gene expression and activation of different intracellular signalling pathways contribute to cell morphogenesis. Rho guanosine triphosphatases (GTPases) are master regulators of the actin cytoskeleton, which affects cell shape and the position of integrins and cadherins, actin cytoskeleton attached cell surface receptors. In addition, Rho GTPases regulate endocytosis, vesicle transport, secretion and gene expression. Not surprisingly, therefore, this family of molecules was found to be particularly important for cell morphogenesis. Key Concepts: Rho GTPases are signal integrators. Rho GTPases have overlapping functions. Rho GTPases regulate cell shape by controlling remodelling of the actin cytoskeleton. Cdc42 regulates different aspects of cell polarity in a cell type‐specific manner. Rho GTPases regulate cell adhesion to the extracellular matrix and to other cells.

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Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

Abstract: Recent studies in Cdc42 knockout mouse embryonic stem (ES) cells and ES-derived

Cited by 138 publications

References 34 publications

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

Rho GTPase Function in Cell Morphogenesis

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