Migrating cells respond to increasing stiffness gradients, an important factor in fibrosis and cancer metastasis. The major mechanisms underlying how stiff environments can be translated into migration signals is not well understood. Focal adhesions are complex, dynamic organelles that enable cells to sense the stiffness of their local environment by linking the actin cytoskeleton to the extracellular substrate. This link at focal adhesions controls a number of signalling pathways, including activation of RhoGTPases. The major underlying mediators that translate high mechanical tension to RhoGTPase activity have yet to be identified. We aim to discover novel tension-sensitive signaling pathways, and understand the mechanisms of respone to tension by developing analogs of the following molecules:
- Vinculin, known as the cell’s molecular clutch, is a prominent linker of focal adhesions to the actin cytoskeleton. Using optogenetic regulation of engineered domains allosterically coupled to vinculing binding sites, I am controlling vinculin-actin interactions. I will control focal adhesion tension and study how this locally controls cell protrusion pathways.
- Vinculin and p130cas undergo dramatic conformational changes due to fluctuations of mechanical strain felt at focal adhesions. Using our novel binder/tag technology, I aim to create a biosensor that will quantify stretch-induced conformational changes and binding interactions of p130cas and vinculin at focal adhesions. I will correlate focal adhesion tension with localized RhoGTPase activity, and use these analogs to create a high throughput assay to identify novel tension-sensitive proteins.
- Durotaxis assays traditionally utilize a stiffness gradient that spans an entire coverslip, but fails to capture the local cell-scale anisotropic stiffness gradients that migrating and metastasizing cancer cells experience in vivo. In order to address this, I have developed a novel topology independent micropillar assay (TIM) that will allow us to investigate the role that local anisotropic traction stresses have on protrusion activity/ cell polarity and local RhoGTPase activity.