Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Actin Cytoskeleton / drug effects
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Actin Cytoskeleton / genetics
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Actin Cytoskeleton / metabolism
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Actin Cytoskeleton / ultrastructure
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Animals
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Aorta / cytology
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Aorta / drug effects
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Aorta / metabolism
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Benzyl Alcohol / pharmacology
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Cattle
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Cell Membrane / chemistry
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Cell Membrane / drug effects
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Cell Polarity / drug effects
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Cells, Cultured
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Cytochalasin D / pharmacology
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Endothelial Cells / cytology
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Endothelial Cells / drug effects
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Endothelial Cells / metabolism*
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Fluorescence Resonance Energy Transfer
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Focal Adhesion Protein-Tyrosine Kinases / antagonists & inhibitors
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Focal Adhesion Protein-Tyrosine Kinases / genetics
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Focal Adhesion Protein-Tyrosine Kinases / metabolism*
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Gene Expression Regulation
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Mechanotransduction, Cellular
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Membrane Fluidity / drug effects*
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Mutation
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Protein Kinase Inhibitors / pharmacology
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Pyrazoles / pharmacology
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Pyrimidines / pharmacology
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Stress, Mechanical
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ras Proteins / genetics
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ras Proteins / metabolism
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rhoA GTP-Binding Protein / genetics
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rhoA GTP-Binding Protein / metabolism*
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src-Family Kinases / genetics
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src-Family Kinases / metabolism*
Substances
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4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine
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Protein Kinase Inhibitors
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Pyrazoles
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Pyrimidines
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Cytochalasin D
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Focal Adhesion Protein-Tyrosine Kinases
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src-Family Kinases
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ras Proteins
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rhoA GTP-Binding Protein
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Benzyl Alcohol