Analyzing external factors affecting cell movement: From substrate rigidity to extra-cellular stress

Date of Completion

January 2011

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Cell movement is a pivotal component for many physiological processes, including wound healing, embyrogenesis, and the progression of cancer. Yet, the mechanisms controlling the cell movement process are poorly understood. While some pathways would be desired to enhance cell movement, such as for wound healing, these same pathways can be undesirable, as seen in the spread of cancer cells. The cell cytoskeleton is the crux of cell movement. While there are intracellular factors that influence the cytoskeleton, external factors can also influence cell movement via the activation of different pathways and proteins. ^ This thesis is composed of a two part study. The first part investigates the effect of substrate rigidity on cell movement via cell retraction mechanisms. Most in vitro studies observe cell dynamics on a rigid surface, such as glass or plastic. However, in vivo, cells often move on top of other cells or on flexible extracellular matrices such as collagen. Cell movement using fish epithelial keratocytes, a fast moving cell type, was compared using gelatin, a flexible substrate, or glass, a rigid substrate. Cell speed, cell morphology, adhesion localization and adhesion strength were used to determine if cells utilize calcium dependent or independent retraction mechanisms on different substrates. Data show cells on a more flexible substrate have weaker initial adhesion strength and utilize calcium dependent and independent retraction pathways.\^ To observe how stress affects cell movement, the second part of this thesis challenged fish keratocytes and SW480, human colon cancer cells, with heat stress. Heat stress resulted in a decrease in cell speed. Cell morphology was also affected by heat shock, resulting in keratocytes losing their classic fan shape and becoming "stickier" or apolar. HSP27, a small heat shock protein, was investigated since previous studies indicated it has actin cytoskeletal interaction. Using co-immunoprecipition and immunofluorescence, HSP27 was shown to be critical for wound closure and to have biochemical interactions with actin.^ These observations further our knowledge of how the cell cytoskeleton is influenced by external factors. This knowledge can assist not only future in vitro studies, but also be applied to the development of new therapeutic approaches. ^

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