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PMID: 27147504 Published · epublish English Journal Article Research Support, Non-U.S. Gov't

Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.

Scientific reports ·Vol. 6 ·2016-00-05 ·Pages 25502

Reinhard NR, van Helden SF, Anthony EC, Yin T, Wu YI, Goedhart J, Gadella TW, Hordijk PL

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.

MeSH Terms
Antigens, CD/genetics,metabolism Bacterial Proteins/genetics,metabolism Biosensing Techniques/methods Cadherins/genetics,metabolism Enzyme Activation Fluorescence Resonance Energy Transfer Gap Junctions Gene Expression Regulation Genes, Reporter Green Fluorescent Proteins/genetics,metabolism Human Umbilical Vein Endothelial Cells/drug effects,enzymology,ultrastructure Humans Luminescent Proteins/genetics,metabolism Models, Molecular Nocodazole/pharmacology Primary Cell Culture Protein Structure, Secondary Signal Transduction Thrombin/pharmacology Tumor Necrosis Factor-alpha/pharmacology rhoA GTP-Binding Protein/genetics,metabolism rhoB GTP-Binding Protein/genetics,metabolism rhoC GTP-Binding Protein/genetics,metabolism
Chemicals
Antigens, CD Bacterial Proteins Cadherins Luminescent Proteins Tumor Necrosis Factor-alpha cadherin 5 yellow fluorescent protein, Bacteria RHOA protein, human Green Fluorescent Proteins Thrombin RHOC protein, human rhoA GTP-Binding Protein rhoB GTP-Binding Protein rhoC GTP-Binding Protein Nocodazole
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Reinhard Nathalie R
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.
van Helden Suzanne F
Sanquin Research, Molecular Cell Biology, Amsterdam, The Netherlands. | Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
Anthony Eloise C
Sanquin Research, Molecular Cell Biology, Amsterdam, The Netherlands. | Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
Yin Taofei
Center for cell analysis and Modeling, University of Connecticut Health Center, Farmington, United States of America.
Wu Yi I
Center for cell analysis and Modeling, University of Connecticut Health Center, Farmington, United States of America.
Goedhart Joachim
University of Amsterdam, Molecular Cytology, Swammerdam Institute for Life Sciences, van leeuwenhoek Centre for Advanced Microscopy, Amsterdam, The Netherlands.
Gadella Theodorus W J
University of Amsterdam, Molecular Cytology, Swammerdam Institute for Life Sciences, van leeuwenhoek Centre for Advanced Microscopy, Amsterdam, The Netherlands.
Hordijk Peter L
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.
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Article Info
Journal
Scientific reports
Abbr.
Sci Rep
ISSN
2045-2322
DOI
10.1038/srep25502
Published
2016-00-05
Epub
2016-00-05
Pages
25502
Language
English
Region
England
NLM ID
101563288
PMCID
PMC4857094
Subset
IM
Grants
NIGMS NIH HHS · R01 GM117061 · United States
External Links
PubMed source DOI: 10.1038/srep25502 PMC Full Text View journal details
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