(I-328) Force-dependent Proximal Biotinylation Surrounding Actin Filaments in Live Cells

To identify force-dependent interactions surrounding actin, we fused TurboID, a biotin ligase that promiscuously biotinylates proximal proteins, with F-tractin, an actin-binding domain of ITPKA, which co-localized with phalloidin labeled actin filaments in cells (Figure 1A). Stable MDCK cell lines expressing TurboID-F-tractin were selected based on homogeneity and low expression of TurboID-F-tractin. The cells were plated on a collagen-coated, thin PDMS membrane and stretched at 50% for 15 minutes in the presence of biotin. These cells were then scraped, lysed, and the biotinylated proteins were purified using streptavidin beads, and analyzed using mass spectrometry. The samples were collected in triplicates. The candidate proteins found in control and stretch conditions were selected based on abundance in the control (non-stretch) and stretch conditions, but were absent in no biotin control. These candidates were ranked according to the statistical significance and the relative abundance of protein in the stretch and control samples (Figure1B, p-value < 0.05). To verify the mass spectrometry results, the relative levels of proteins (Western blot analysis) and sub-cellular localization (immunofluorescence analysis) were also tested. Lastly, a plasmid containing GFP tagged with each candidate protein was designed and constructed using Gibson Assembly, and transfected into MDCK cells to test its force-sensitivity in live cells.