RUI: MCB: the effect of stretch on giant cytoskeletal protein structure/function

James Madison University, Harrisonburg VA

Investigators

Abstract

Cells live in a world full of motion and physical strain, and therefore they must have mechanisms to react to these stresses. One platform cells use to sense and respond to stretch is the cytoskeleton. This meshwork of multiple different proteins gives a cell its shape, yet is constructed in a way that allows flexibility and motion. This research is focused on the role that the cytoskeletal protein obscurin plays in stretch response and recognition. Obscurin acts like a tether, connecting far-away segments of the cell to each other. Due to its shape, obscurin has the capacity to expand and contract. There are two possibilities of how obscurin could move like this. Obscurin could behave like a rope, and only resist stretch when significantly elongated, or obscurin could behave like a spring, and resist an ever-increasing amount of force as it is stretched. Obscurin also can propagate biochemical signals, and there is circumstantial evidence that this function could be activated by stretch. The PI will test both facets of obscurin function - stretch response and signaling. Together, these studies will provide insight into how cells passively and actively respond to physical stretch. This work will be conducted primarily by undergraduate students, in an effort to train the next generation of scientists. The data accrued here will be incorporated into a free education website, where others who do not have access to significant research support can also learn the technical skills of how to do this kind of research. Additionally, the PI will develop a scientific ethics curriculum for undergraduates. Specifically, the investigator will study how obscurin reacts to physical stretch using both cellular and in vitro models. Obscurin is composed of multiple stand-alone domains. Many of these individual domains have been characterized extensively. In Aim 1, the PI will characterize how these domains act in groups of two or three. Using protein NMR techniques, the cross-talk between neighboring domains will be measured. Small angle X-ray scattering (SAXS) provide a dynamic model of obscurin's overall shape. These experimental approaches will be complimented through computer simulations to test how these multi-domain systems respond to stretch. Together, these experiments will detail how obscurin resists force. In Aim two, the PI will test what effect obscurin has on the whole cell when the cell is stretched. Cells with and without obscurin will be stretched, and the biochemical consequences of obscurin's presence in these conditions will be analyzed. Together, these two aims will help define how obscurin behaves as a stretch resistor, and will answer the question of whether or not obscurin is a mechanosensor.