This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Vascular endothelial cells (ECs) are constantly subjected to cyclic stretch due to pulsatile blood pressure. In straight, unbranched arteries, ECs are elongated with their major axes oriented with that of the arterial vessel, i.e. perpendicular to the direction of circumferential stretch. Fibers aligned perpendicular to stretch bear less tension than when they are aligned parallel to stretch. Thus, perpendicular orientation of the stress fibers serves to reduce the stretch-induced tension and thus minimize the intracellular mechanical strain induced by uniaxial stretch. Preliminary experiments have shown that an increase of Rho activity in ECs enhances the perpendicular orientation of stress fibers to stretch and that the inhibition of Rho activity causes the stress fibers to be oriented parallel to the direction of stretch [1]. These results led us to propose that Rho GTPase plays a central role in regulating the stretch-modulation of intracellular mechanical strain through the control of stress fiber re-organization. Experimental testing of the model requires an elucidation of the role of cell viscoelastic properties, due in part to the cytoskeleton, in the transmission and transduction of surface stresses. Transduction of the stresses will lead to Src kinase signaling [2, 3], an event that we can monitor in real time through a genetically encoded Src kinase FRET sensor [3]. We will use the proposed LAMMP Spatially Modulated Microbeams (SMM) system to apply stresses through laser tweezer-microbeads interactions in order to study the relationship between cell mechanics (surface stresses, strain and strain rates) and stress transduction by monitoring the mechanically induced Src signaling. The experiments will be conducted on Human Umbilical Vein Endothelia Cells (HUVEC or EC) transfected with a GFP-fused mitochondrial protein or microinjected with fluorescent microbeads, and also transfected with the Src sensor in order to determine the molecular and cellular correlates of the intracellular mechanics.
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