title: Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.
abstract: Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells.
authors.affiliation: University of Amsterdam, Molecular Cytology, Swammerdam Institute for Life Sciences, van leeuwenhoek Centre for Advanced Microscopy, Amsterdam, The Netherlands.|Sanquin Research, Molecular Cell Biology, Amsterdam, The Netherlands.|Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
authors.forename: Nathalie R
authors.initials: NR
authors.lastname: Reinhard
authors.affiliation: Sanquin Research, Molecular Cell Biology, Amsterdam, The Netherlands.|Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
authors.forename: Suzanne F
authors.initials: SF
authors.lastname: van Helden
authors.affiliation: Sanquin Research, Molecular Cell Biology, Amsterdam, The Netherlands.|Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
authors.forename: Eloise C
authors.initials: EC
authors.lastname: Anthony
authors.affiliation: Center for cell analysis and Modeling, University of Connecticut Health Center, Farmington, United States of America.
authors.forename: Taofei
authors.initials: T
authors.lastname: Yin
authors.affiliation: Center for cell analysis and Modeling, University of Connecticut Health Center, Farmington, United States of America.
authors.forename: Yi I
authors.initials: YI
authors.lastname: Wu
authors.affiliation: University of Amsterdam, Molecular Cytology, Swammerdam Institute for Life Sciences, van leeuwenhoek Centre for Advanced Microscopy, Amsterdam, The Netherlands.
authors.forename: Joachim
authors.initials: J
authors.lastname: Goedhart
authors.affiliation: University of Amsterdam, Molecular Cytology, Swammerdam Institute for Life Sciences, van leeuwenhoek Centre for Advanced Microscopy, Amsterdam, The Netherlands.
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.affiliation: University of Amsterdam, Molecular Cytology, Swammerdam Institute for Life Sciences, van leeuwenhoek Centre for Advanced Microscopy, Amsterdam, The Netherlands.|Sanquin Research, Molecular Cell Biology, Amsterdam, The Netherlands.|Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Hordijk
mesh_terms: D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D015820:Cadherins / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D017629:Gap Junctions; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015739:Nocodazole / Q000494:pharmacology; D061251:Primary Cell Culture; D017433:Protein Structure, Secondary; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D001426:Bacterial Proteins; D015820:Cadherins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D015739:Nocodazole; D013917:Thrombin; D014409:Tumor Necrosis Factor-alpha; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein; D000099152:Cadherin 5; C054562:yellow fluorescent protein, Bacteria; C066737:RHOA protein, human; C066690:RHOC protein, human