The long term objectives of the proposed research are to understand how the proteins responsible for motility in cells are organized into contractile units and what factors control the formation and function of these units. The specific goals seek to use fluorescently labeled contractile proteins to investigate how stress fibers, cleavage rings and myofibrils are formed in living cells; to examine the interactions of four different actin-binding proteins with stress fibers and myofibrils in living and permeabilized cells; and to extend current knowledge of stress fiber and cleavage ring ultrastructure. The major focus of our proposal is on microinjection studies using fluorescently labeled proteins. Myosin, actin, alpha-actinin and tropomyosin will be prepared from both muscle and non-muscle sources, labeled with different fluorescent dyes and microinjected into a variety of cultured epithelial and fibroblastic cells as well as embryonic myocytes in culture and in situ. Actin will be injected as profilactin and in the uncomplexed monomer form, G-actin. The length of time required for incorporation and the longevity of the incorporated protein will be compared for the same protein prepared from different sources and using different fluorescent dyes. The ability of the labeled proteins to cycle as stress fibers, cleavage rings and myofibrils assemble and disassemble will be examined using low-light level cameras coupled with video-enhanced microscopy. Photo-bleaching experiments using an argon laser will be used to measure the recovery times of labeled proteins into stress fibers, cleavage furrows and myofibrils and to determine whether or not addition occurs as a treadmilling process. Two actin-binding proteins, vitamin D binding protein and brain capping protein, will be reacted with isolated myofibrils and permeabilized non-muscle cells to determine whether they interfere with the binding of exogenous contractile proteins to these systems. In addition, spectrin and filamin will be purified and labeled with fluorescent tags to determine if they bind to the permeabilized systems. Subsequently, these four proteins will be injected into living cells so that their interactions with stress fibers, cleavage furrows and myofibrils can be examined. The ultrastructure of stress fibers and cleavage rings will be studied using gold labeled antibodies against contractile proteins and actin-binding proteins such as filamin. The information gained in these studies will provide basic knowledge of motility in normal cells and provide a framework against which to compare motility in cells involved in processes such as wound healing or cancer in which transformed cells lack stress fibers.
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