RhoA and Membrane Fluidity Mediates the Spatially Polarized Src/FAK Activation in Response to Shear Stress

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/cm2) 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.

Protein Interactions at Endothelial Junctions and Signaling Mechanisms Regulating Endothelial Permeability

“…These signaling pathways in turn orchestrate a coordinated cellular response resulting in reorganization of the actin cytoskeleton, redistribution of intracellular tension, and a shift in phosphorylation of VE-cadherin and associated catenins 142, 167–169 . Using a recently developed biosensor that measures actomyosin-mediated tension across VE-cadherin adhesion and PECAM-1, it was shown that shear stress applied to an endothelial monolayer reduces tension across VE-cadherin adhesion concomitant with a decrease in total cell-cell force 142, 170 . Thus, it appears that the distribution of intracellular tension is tightly regulated in response to external mechanical forces thus allowing AJs to align in the direction of flow.…”

“…This is evident by the finding that they induce differential phosphorylation of VE-cadherin and p120-catenin 165, 166, 188–190 . Some mediators, such as thrombin, function through phosphorylating VE-cadherin at Tyr 658 by c-Src 170, 172 or p120 catenin at Ser879 by protein kinase C (PKC)α resulting in decreased binding of VE-cadherin to p120-catenin 185, 189 . Indeed, leakage of tumor vessels is associated with c-Src-dependent phosphorylation of VE-cadherin and β- and p120-catenin proteins 191 .…”

Periodic Mechanical Stress Induces Extracellular Matrix Expression and Migration of Rat Nucleus Pulposus Cells Through Src-GIT1-ERK1/2 Signaling Pathway

“…One study has reported that Src plays crucial roles in shear stress-induced cellular processes [23]. A report published by Hum et al revealed that mechanical loading may induce the formation of a Src/Pyk2/MBD2 complex in the nucleus, that functions to suppress anabolic gene expression [24].…”

Intravital microscopy of cancer: New insights into the spatiotemporal dynamics of the disease

“…For instance, subcellular ATP FRET-biosensors are used to visualize intracellular energy transport following inhibition of glycolysis (7), while oxygen FRET-based biosensors can monitor intracellular oxygen transport and consumption (8). Wang and colleagues have developed a simultaneous multi-parametric analysis of distinct FRET biosensors including Src, RhoA and FAK and, thereby, demonstrated their interdependent regulation in response to mechanoreciprocity, which is known to drive tissue dysplasia and metastasis in a variety of cancers (9,10). In addition, the development of Raichu Rho GTPase FRET-biosensors has significantly improved the understanding of actomyosin cytoskeletal remodeling, cell proliferation and motility in cancer (11).…”