RhoA and membrane fluidity mediates the spatially polarized Src/FAK activation in response to shear stress.
While Src plays crucial roles in shear stress-induced cellular processes, little is known on the spatiotemporal pattern of high shear stress (HSS)-induced Src activation. HSS (65 dyn/cm(2)) was applied on bovine aortic endothelial cells to visualize the dynamic Src activation at subcellular levels utilizing a membrane-targeted Src biosensor (Kras-Src) based on fluorescence resonance energy transfer (FRET). A polarized Src activation was observed with higher activity at the side facing the flow, which was enhanced by a cytochalasin D-mediated disruption of actin filaments but inhibited by a benzyl alcohol-mediated enhancement of membrane fluidity. Further experiments revealed that HSS decreased RhoA activity, with a constitutively active RhoA mutant inhibiting while a negative RhoA mutant enhancing the HSS-induced Src polarity. Cytochalasin D can restore the polarity in cells expressing the active RhoA mutant. Further results indicate that HSS stimulates FAK activation with a spatial polarity similar to Src. The inhibition of Src by PP1, as well as the perturbation of RhoA activity and membrane fluidity, can block this HSS-induced FAK polarity. These results indicate that the HSS-induced Src and subsequently FAK polarity depends on the coordination between intracellular tension distribution regulated by RhoA, its related actin structures and the plasma membrane fluidity.
1] Department of Biomedical Engineering, Dalian University of Technology, Dalian, Liaoning Province, 116024, P. R. China [2] Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Bo
B
Liu
Department of Bioengineering, University of California at San Diego, CA 92093, USA.
Shaoying
S
Lu
Department of Bioengineering, University of California at San Diego, CA 92093, USA.
Ying-li
YL
Hu
1] Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA [2] Biomaterials and Live Cell Imaging Institute, Chongqing University of Science and Technology, Chongqing, 401331, P. R. China.
Xiaoling
X
Liao
Department of Bioengineering, University of California at San Diego, CA 92093, USA.
Mingxing
M
Ouyang
1] Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA [2] Department of Integrative and Molecular Physiology, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA [3] Department of Bioengineering, University of California at San Diego, CA 92093, USA.
Yingxiao
Y
Wang
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019905:Benzyl Alcohol / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D040542:Mechanotransduction, Cellular; D008560:Membrane Fluidity / Q000187:drug effects*; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013314:Stress, Mechanical; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D015638:Cytochalasin D; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein; D019905:Benzyl Alcohol