Project SummaryDuring embryogenesis or wound healing, neighboring cells maintain contact and migrate collectively, thoughthe roles of cell-cell adhesion during collective cell migration is poorly defined. Due to constant pulling andpushing between migrating neighboring cells, we hypothesize that mechanical forces regulate theinteraction between the cell-cell adhesion complex and the actin cytoskeleton, and therefore, theadhesive strength. To identify force-sensitive protein complexes at cell-cell junctions, our innovativebiochemical analysis combines in situ proximal biotin labeling with a cell stretch device that promotes theformation of force-sensitive complexes. By fusing ?-catenin with a promiscuous biotin ligase, any proximalproteins of ?-catenin will be biotinylated. The force-dependent change in the biotinylation profile is an indicationof altered protein complexes. Our preliminary study demonstrates that ?-catenin and myosin IIA are likelyinteracting in a force-dependent manner. While the current approach is suited for the candidate screening offorce-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 thepresence 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 limitthe quantity of protein samples. Our goal of this proposal is to re-design and scale-up the currentprotocol to isolate the quantity of purified proteins sufficient for mass spectrometry and identify theforce-sensitive complex surrounding ?-catenin. We will fabricate cell stretch chambers based on a siliconsheet as a substrate to culture cells. Using this device, we will optimize the mechanical stimulation (thefrequency, the magnitude and the duration of substrate stretch) based on cell morphology, the organization ofthe actin cytoskeleton, and the extent of biotinylation. Using the newly designed cell stretcher and massspectrometry analysis, we will determine a comprehensive list of the force-sensitive molecules surrounding ?-catenin that is essential for understanding of mechano-transduction.
|Effective start/end date||7/1/16 → 4/30/18|
- National Institutes of Health: $70,815.00