Mechano-Sensing and the Integration of Cytoskeletal Function in Moving Cells

University Of Connecticut, Storrs CT

Investigators

Abstract

Cell movement depends on the integration of several cytoskeletal functions such that protrusion and adhesion formation occur at the front, while adhesion disassembly and retraction are localized to the rear. Although much has been learned about the molecular basis of these processes, it is not clear how they are integrated to produce movement at the cellular level, and represents a major gap in understanding of cell motility. The study of fish epithelial keratocytes can provide some valuable insight into this question because they exhibit a simple, semi-circular shape, and rapid highly directed mode of movement. In addition their movement is known to be regulated by the activation of stretch-activated calcium channels (SAC''s) that act as mechano-sensors to trigger retraction at the rear, in response to increases in cytoskeletal tension. Previous NSF supported work from the Lee laboratory showed that SAC mediated calcium transients induce spatially coordinated increases in cytoskeletal force generation that together with a rise in intracellular calcium ([Ca2+]i), facilitate adhesion disassembly and retraction at the rear, while promoting adhesion formation and protrusion at the front. These data suggest that the local mechano-sensory response of adhesions to force predisposes adhesions at the rear to Ca2+ dependent disassembly, while preferentially strengthening newer adhesions at the front. Intellectual Merit: The project will test the hypothesis that cooperative mechano-sensing between SAC''s and adhesion complexes integrates local regulation of cytoskeletal functions to produce movement at the cellular level. The studies will utilize a combination of fluorescence imaging and force measurement techniques in single moving keratocytes expressing a variety of fluorescently tagged adhesion proteins. Specific aim 1 will quantify the effect of altering [Ca2+]i on adhesion composition and turnover, at different cellular locations. Specific aim 2 will elucidate the molecular mechanism(s) by which force and [Ca2+]i coordinately regulate adhesion turnover. Specific aim 3 will quantify the relationship between force and [Ca2+]i induced changes in adhesion turnover with their mechanical coupling to the substratum. Broader impacts of the project: Dr. Lee''s research program attracts many students to science because of the inherent beauty of living cells that is revealed to them through microscopic imaging. The project is interdisciplinary in nature, and so provides a rich and varied learning experience for undergraduates and graduate students. As a result students learn how to utilize knowledge and combine techniques not only from within cell biology, but across disciplines. The ability to work at the interface of different scientific disciplines leaves students well-prepared for a wide range of scientific careers. Students report their findings at lab meetings, journal club, departmental seminars, at national meetings and in the form of publications in leading journals. Many of Dr Lee''s students are women or from underrepresented minority groups. Each year Dr. Lee and her female students participate in a one-day workshop designed to attract high school girls to the fields of science and engineering.