Date of Completion

6-18-2014

Embargo Period

6-17-2014

Advisors

Dr. David Knecht, Dr. Adam Zweifach

Field of Study

Molecular and Cell Biology

Degree

Master of Science

Open Access

Open Access

Abstract

Collective migration is a critical biological process that involves the coordinated migration of a group of cells connected via adherens junctions. These cell-cell adhesions are regulated by intracellular and extracellular calcium, and require controlled turnover in order to maintain the structural integrity of migrating cell sheets. In single fish keratocytes, intracellular calcium signaling via stretch-activated calcium channels facilitates migration by regulating cytoskeletal contractility and cell-substratum adhesion turnover. The role of calcium signaling in collectively migrating keratocyte sheets remains unknown. In this study, we investigate the effects of modulating the frequency of calcium transients in goldfish keratocyte sheets, using EGTA and Thapsigargin, on sheet morphology and cohesiveness. We also use varying serum concentrations and Gadolinium to investigate the role of serum and stretch-activated calcium channels, respectively, in the production of calcium transients and in the maintenance of sheet cohesiveness. We find that calcium transient frequencies diminish in the presence of EGTA, Thapsigargin, and Gadolinium, or in serum-free medium. These results suggest that, in keratocyte sheets, calcium transients occur via stretch-activated channels and require both extracellular calcium and intracellular calcium stores, as well as serum. Sheet area and cohesiveness decrease in Thapsigargin-treated cells, and sheet cohesion is reduced in cells treated with EGTA and serum-free medium. This suggests that keratocyte sheets require calcium transients for maintaining cell-cell adhesions. We propose that serum and stretch-activated channels work in concert to regulate fluctuations in cytoskeletal contractility, thus facilitating the controlled turnover of adherens junctions needed to maintain cell-cell adhesions during collective migration.

Major Advisor

Dr. Juliet Lee

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