Abstract
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.
Highlights
It is clear that mechanical forces play vital roles in shaping the normal functions of all tissues and organs of human beings [1]
As soon as the local mechanical stress (17.5 Pa, step function) was applied, the Rac GTPase was activated within 300 ms, as evidenced by the significant changes in fluorescent resonance energy transfer (FRET) at the cell peripheries (Fig. 2A, C)
The mechanism of mechanotransduction, i.e., how mechanical forces are converted into biochemical activities inside the cell is a central question in cell biology
Summary
It is clear that mechanical forces play vital roles in shaping the normal functions of all tissues and organs of human beings [1]. The main thrust of these models is that mechanotransduction, similar to the soluble factor induced signal transduction, initiates at the cell membrane by inducing local conformational changes or unfolding of membrane-bound proteins at the site of a local force, followed by a cascade of diffusion and translocation processes for downstream signaling. This is consistent with the theory of the classical continuum mechanics of St. Venant’s principle that a local force must cause only a local deformation. We examined whether Rac can be directly activated by stress independent of Src
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