Project 2.1. Develop fluorescent biosensors to quantify the spatio-temporal dynamics of protein activity in living cells during the application of mechanical forces, using novel designs with enhanced sensitivity and reduced physiological perturbation. Specific new biosensors requested by CISMM collaborators are produced using biosensor designs previously validated by our laboratory. We also test new biosensor designs that reduce physiological perturbation by reporting activation of endogenous proteins. Protein fragments that bind to specific states of target molecules will be derivatized with fluorescence resonance energy transfer (FRET) ‘binding antennae’, pairs of fluorescent proteins configured to change FRET fluorescence intensity upon binding of the protein fragment to the target. Our existing GTPase biosensors are also re-engineered using recently discovered viral peptide to enhance sensitivity and ease of use.
Recently, the Tzima lab showed that localized forces on PECAM-1 elicit global mechanotransduction. [Collins et al 2012]. Using the magnetic tweezers system and sophisticated biosensors designed by the Hahn lab, we showed that force on PECAM-1 increases cellular stiffening via activation of phosphatidylinositol 3-kinase (PI3K) (Fig. A, B). This activation of PI3K downstream of PECAM-1 promoted cell-wide activation of integrins and the small GTPase RhoA. In complementary studies, we showed that the mechanical force of shear stress induces polarized Rac activation (again using biosensors from the Hahn lab) via mechanoactivation of the Rac GEFs, Tiam1 and Vav2 (Liu et al JCB 2013)