Biochemical analysis of force-sensitive responses using a large-scale cell stretch device

Abstract

Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses. Using proximal biotinylation (BioID) analysis or phospho-specific antibodies, we detected force-sensitive biochemical changes in cells exposed to prolonged cyclic substrate stretch. For example, using promiscuous biotin ligase BirA* tagged α-catenin, the biotinylation of myosin IIA increased with stretch, suggesting the close proximity of myosin IIA to α-catenin under a force bearing condition. Furthermore, using phospho-specific antibodies, Akt phosphorylation was reduced upon stretch while Src phosphorylation was unchanged. Interestingly, phosphorylation of GSK3β, a downstream effector of Akt pathway, was also reduced with stretch, while the phosphorylation of other Akt effectors was unchanged. These data suggest that the Akt-GSK3β pathway is force-sensitive. This simple cell stretch device enables biochemical analysis of force-sensitive responses and has potential to uncover molecules underlying mechano-transduction.

Spatial proximity of proteins surrounding zyxin under force-bearing conditions

“…Using a large-scale cell stretch device (Renner et al, 2017), biotinylated proteins were then biochemically isolated from cells plated on control and stretched membranes (60% uniaxial stretch for 30 minutes in the presence of 50 μM biotin) and analyzed using label-free mass spectrometry (Figure 1B and C, see the complete list of proteins in Supplemental Data). To identify highly biotinylated proteins under force-bearing conditions with minimal deviations among the triplicate samples, the ratios of relative abundance of identified biotinylated proteins in control versus stretch conditions were plotted against the standard deviation of the ratios (Figure 1C, and Supplemental Data, "ratio" tab).…”

“…Using a large-scale cell stretch device ( Renner et al. , 2017 ), biotinylated proteins were then biochemically isolated from cells plated on control and stretched membranes (60% uniaxial stretch for 30 min in the presence of 50 μM biotin) and analyzed using label-free mass spectrometry ( Figure 1, B and C ; see the complete list of proteins in the Supplemental Data).…”

“…Cells were stretched for 10 minutes in the presence of 50 μM biotin, then stained with anti-v5 antibody and streptavidin (Figure 1A). For the large-scale analysis for mass spectrometry analysis, the cells were plated on the thin collagen-coated PDMS membranes attached to custom acrylic plate and inserted into a square cell culture dish (see details in Renner et al, 2017). After 24 hours, the membranes were uniaxially stretched to 60 % for 30 minutes in the presence of 50 μM biotin (Sigma-Aldrich).…”