Moving cells show a pronounced rearward flow of membrane and cytoskeletal structures from the marginal lamellipodia through the lamellar cortex. The hypothesis to be explored is that this flow is an essential mechanism of cell locomotion and that it is driven, in part, by an actomyosin-like contraction of a more centrally placed lamellar cortex. This will be studied using computer- enhanced video microscopy in combination with immunocytochemistry, single-cell electron microscopy, and fluorescence marking to examine the movement and the three dimensional organization and flow of actin filaments and bundles in protruding lamellipodia. The mechanisms of the flow of the dorsal cortical microfilament sheath of the lamella will be examined using specific markers, photobleaching, and myosin II competitors. The recent discovery that stress fibers form in concert with cortical flow will be extended to examine the mechanism of stress fiber elongation and the development of a sarcomeric organization of cytoskeletal proteins in fibers. The rebuilding of the cortex and stress fibers after elimination by cytochalasin will be examined. These studies will advance the field of cell motility by contributing to understanding of the mechanism of cellular migration and its role in the morphogenesis of matrix and tissues. %%% Cellular motility is a fundamental and critical property of virtually all organisms. This project focuses on "ameboid" or "crawling" motility, in which cells on a solid surface extend projections out in the direction in which they are moving and appear to "pull themselves up" to those projections. This form of cellular motility occurs in virtually all animals (invertebrate and vertebrate), and is critical for morphogenesis during development, regeneration and tissue remodelling after injury, host defense mechanisms, and other vital functions, and also occurs in cells of other phyla (e.g., protozoa, slime molds). The approach to be used is to correlate high resolution video-enhanced microscopic observations on the motility of living cells with both ultrastructural observations at the EM level on the same cell and with immunocytochemical localization of specific proteins also on the same cell. Very few other laboratories in the world use this difficult but highly informative approach. These studies will contribute to our understanding of the molecular organization and mechanism of the machinery of cell motility.
