RUI: Probing obscurin's role as a mechanotransducer

Some cells must migrate throughout the body - this is how wounds heal and how organs develop in the correct spot. However, cells that migrate when they shouldn’t are a hallmark of cancer metastasis. Surprisingly little is known about how human cells decide to migrate. This project will investigate a protein named “obscurin” that prevents cells from migrating within the body. When obscurin is missing or mutated, cells migrate more. Cancer cells often get rid of obscurin, which partially explains why they metastasize. It has also been discovered that obscurin is stretched taut in cells. This project will explore the relation between obscurin being under tension and obscurin’s control of migration, and will test the idea that two functions are linked; that obscurin is normally under tension, which signals cells not to migrate; versus when obscurin is not under tension - when the cell is free-floating or is on a Jello-like surface- that osbcurin signals the cell to migrate. We will study these two aspects of obscurin function - tension and motility signaling- to see if obscurin tension and cell surroundings control cell motility. The work will be carried out by undergraduate students, and the results will be present both in scientific papers, and as outreach activities at local farmers’ markets and festivals so that the public can interact with this work. This project will test the hypothesis that the physical environment of the cell dictates the conformation of obscurin, and this in turn regulates cellular motility. The hypothesis will be tested through 3 Aims: Aim 1 tests whether obscurin tension and obscurin activity is the result of the physical environment of the cell, by means of a fluorescence-based tension sensor placed in the middle of obscurin in various cellular conditions, such as plates with very low elastic moduli. Aim 2 asks whether obscurin is activated in an accordion-like model, via attachment of long tails on either side of the obscurin-signaling domains to attempt to occlude obscurin target binding, followed by biochemical and cellular assays. If obscurin activity is blocked by these obscuring flanking domains, it will infer that these domains can normally fold to occlude obscurin-target binding. Aim 3 will test whether the conformation of the obscurin C-terminus changes with physical environment, and thereby affects signaling capability. Here, fluorophores will be attached at various points within the tail domain to measure distances between domains, both in cells under tension, and in lysed cells. The results should provide strong evidence for the mechanism by which obscurin shape and tension dictates binding capacity, and its function in mediating cellular dynamic response to dynamic changes in cellular tension. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.