title: Plasma membrane restricted RhoGEF activity is sufficient for RhoA-mediated actin polymerization.
abstract: The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands.
authors.forename: Jakobus
authors.initials: J
authors.lastname: van Unen
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands.
authors.forename: Nathalie R
authors.initials: NR
authors.lastname: Reinhard
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06032-6406.
authors.forename: Taofei
authors.initials: T
authors.lastname: Yin
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06032-6406.
authors.forename: Yi I
authors.initials: YI
authors.lastname: Wu
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands.
authors.forename: Marten
authors.initials: M
authors.lastname: Postma
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands.
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands.
authors.forename: Joachim
authors.initials: J
authors.lastname: Goedhart
mesh_terms: D000199:Actins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D002467:Cell Nucleus; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D064067:Rho Guanine Nucleotide Exchange Factors; D014157:Transcription Factors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
chemical_list: D000199:Actins; C478036:MRTFB protein, human; D064067:Rho Guanine Nucleotide Exchange Factors; D014157:Transcription Factors; C066737:RHOA protein, human; D020742:rhoA GTP-Binding Protein