Rapid Activation of Rac GTPase in Living Cells by Force Is Independent of SrcReportar como inadecuado

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It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer FRET method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP enhanced cyan fluorescent protein as a donor and YPet a variant yellow fluorescent protein as an acceptor of the Rac biosensor. The applied stress induced rapid activation less than 300 ms of Rac at the cell periphery. In contrast, platelet derived growth factor PDGF induced Rac activation at a much later time >30 sec. There was no stress-induced Rac activation when a mutant form of the Rac biosensor RacN17 was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src activation. These findings suggest that signaling pathways of mechanical forces via integrins might be fundamentally different from those of growth factors.

Autor: Yeh-Chuin Poh , Sungsoo Na , Farhan Chowdhury, Mingxing Ouyang, Yingxiao Wang , Ning Wang

Fuente: http://plos.srce.hr/


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