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Role of the cytoskeleton and substratum in cell topographic guidance Oakley, Carol

Abstract

Topographic guidance refers to reactions of cells with the topography of their substratum and includes alterations in cell shape, orientation and polarity of movement. Current hypotheses focus on the cytoskeleton although most observations have been limited to cells that were already oriented with their substratum topography. In this thesis I addressed this shortcoming, tested the current theories of contact guidance and hypothesized that there may be a prime or principal cytoskeletal determinant of fibroblast orientation on micromachined grooves. I predicted that the principal cytoskeletal determinant of contact guidance would be the first cytoskeletal component to become aligned with the topography, would be most sensitive to size and arrangement of the topography and would be able to exert its influence independently. Furthermore, if the principal determinant was also the sole determinant, then contact guidance would not occur in its absence. Finally, the principal component would operate in other cell types. The hypotheses and predictions were tested directly by examining the development of cytoskeletal alignment in relation to overall cell shape and alignment as fibroblasts spread on micromachined grooved substrata. Time-lapse cinematography, conventional and confocal epifluorescence microscopy and computer-assisted morphometry indicated that microtubules were the first cytoskeletal element to become aligned and microtubule alignment preceded alignment of the cells themselves. Cell spreading and alignment in the presence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin) on micromachined grooves that differed in size demonstrated that the integrity of either the microtubule or actin system could be sufficient to produce alignment with an appropriate substratum topography but cells deficient in microtubules required significantly more time to accomplish these behaviours. These experiments demonstrated that microtubules were the principal but not the sole cytoskeletal determinant of contact guidance on micromachined grooves. Microtubules responded faster and to smaller topographic features than microfilaments but unless the topography elicited alignment of both the microtubule and actin systems, the influence of microtubules upon cell alignment was tempered. I concluded that the substratum topography could selectively or differentially effect alignment of the microtubule and actin systems so that cell alignment and migration could be enhanced or retarded.

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