During embryogenesis or wound healing, neighboring cells maintain contact and migrate collectively, though the roles of cell-cell adhesion during collective cell migration is poorly defined. Due to constant pulling and pushing between migrating neighboring cells, we hypothesize that mechanical forces regulate the interaction between the cell-cell adhesion complex and the actin cytoskeleton, and therefore, the adhesive strength. To identify force-sensitive protein complexes at cell-cell junctions, our innovative biochemical analysis combines in situ proximal biotin labeling with a cell stretch device that promotes the formation of force-sensitive complexes. By fusing ?-catenin with a promiscuous biotin ligase, any proximal proteins of ?-catenin will be biotinylated. The force-dependent change in the biotinylation profile is an indication of altered protein complexes. Our preliminary study demonstrates that ?-catenin and myosin IIA are likely interacting in a force-dependent manner. While the current approach is suited for the candidate screening of force-sensitive proteins, the application of proteomic screening to this approach will be transformative, because the proteomic screening will reveal the total composition of ?-catenin associated proteins in the presence or absence of external forces, a critical first step in deciphering the molecular basis of mechano- transduction. However, the key limitation of the current protocol is that the small cell stretch chambers that limit the quantity of protein samples. Our goal of this proposal is to re-design and scale-up the current protocol to isolate the quantity of purified proteins sufficient for mass spectrometry and identify the force-sensitive complex surrounding ?-catenin. We will fabricate cell stretch chambers based on a silicon sheet as a substrate to culture cells. Using this device, we will optimize the mechanical stimulation (the frequency, the magnitude and the duration of substrate stretch) based on cell morphology, the organization of the actin cytoskeleton, and the extent of biotinylation. Using the newly designed cell stretcher and mass spectrometry analysis, we will determine a comprehensive list of the force-sensitive molecules surrounding ?- catenin that is essential for understanding of mechano-transduction.
Tissues and organs undergo constant physical perturbations. Individual cells respond to mechanical forces to maintain tissue integrity, but the molecular mechanisms used by cells to react to such forces are not well defined. Using innovative techniques to detect force-dependent protein-protein interactions, our goal is to identify the complete set of force-induced molecular complexes at cell junctions. Understanding the regulation of cell-cell interactions should reveal, for example, how carcinoma cells break free from their home tissues and metastasize to tissues elsewhere, and may provide targets for novel therapeutics.
| Cheah, Joleen S; Yamada, Soichiro (2017) A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat. Biochem Biophys Res Commun 493:1522-1527 |
| Renner, Derrick J; Ewald, Makena L; Kim, Timothy et al. (2017) Biochemical analysis of force-sensitive responses using a large-scale cell stretch device. Cell Adh Migr 11:504-513 |
| Lee, Eliot; Ewald, Makena L; Sedarous, Mary et al. (2016) Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission. Biochem Biophys Res Commun 478:1640-6 |
