Measuring Vinculin Tension in Focal Adhesions Reveals Force-Dependent ProteinProtein Interactions
Andrew S. LaCroix1, Brenton D. Hoffman, Ph.D.1
1Duke
University, Durham, NC
Cells convert mechanical stimuli into biochemical signals through a poorly understood process
called mechanotransduction. Aberrant mechanical signaling has been linked to diseases such
as atherosclerosis, muscular dystrophy, and cancer. Cells adhere to their extracellular matrix
through complex, multi-protein, force-responsive structures called focal adhesions (FAs). Over
150 proteins are known to localize to FAs, but the protein-protein interactions mediating this
force-sensitivity have yet to be revealed. The recent development of a FRET-based molecular
tension sensor allows me to measure the average tension across vinculin in individual FAs. We
hypothesize that vinculin under differential tension facilitates the recruitment of distinct FA
proteins, mediating downstream signaling pathways. By combining measurements of vinculin
tension with immunofluorescence staining for proteins of interest, we are able to determine
which FA proteins accumulate at FAs in either a force-dependent or a vinculin-dependent
manner. While we have probed ~30 proteins to date, here we describe focal adhesion kinase
(FAK) as a representative case. FAK is preferentially phosphorylated at the Y925 residue in FAs
where vinculin is under high force. Since vinculin tension has been shown to be higher at the
leading edge of migrating cells, this finding suggests FAK phosphorylation may be one key
mechanoresponse to vinculin tension in cell protrusions. Comparisons of full length tension
sensor with a tail-less version indicates the tail domain of vinculin is involved in this
phosphorylation switch. Combining similar analyses with live cell imaging could be used to
investigate dynamic relationships among FA proteins, enhancing our understanding of
mechanotransduction.