The goal of the proposed work is to provide information that will help in the understanding of how cells move and change shape. Cell motility is necessary for such physiologic processes as phagocytosis, platelet response during blood coagulation, wound healing, and neurologic development. The proposed research will explore three aspects of this problem, using a combination of biochemical and biophysical techniques. The first project will define how the structure and reactivity of proteins already thought to be needed for motility are affected by the intracellular biochemical changes that occur as the result of stimuli that cause cell motion. Specifically, the effects of calcium ions and polyphosphoinositides (PPI's) on the polymerizable protein actin and its regulatory proteins gelsolin and profilin will be examined using optical methods that have been developed to quantify the length and concentration of filaments formed by actin. Understanding the link between cell signalling and motility is important for understanding the defects that cause impaired or uncontrolled movement and proliferation. Second, the material properties of all three of the major filamentous proteins (F-actin, microtubules, and intermediate filaments) that are thought to endow a cell with mechanical strength will be measured. These studies will directly compare, for the first time, the physical properties of all three types of filaments in order to ascertain the special features of each type, which may suggest specialized roles for different filaments within the cytoplasm. These studies will employ specialized instruments commonly used in polymer physics to measure the strength and elasticity of various filament networks as molecular properties such as length or stiffness of filaments, or the presence of links between filaments are varied by different regulatory proteins. This work may help identify which proteins or metabolites cause particular morphologic changes observed in living cells. The third project will attempt to identify how the forces causing motion in non-muscle cells are generated. Currently there are several different theories describing how such forces could be generated, but experimental data to distinguish among them are lacking. This work will involve forming simple biochemical models for cells which contain actin or other proteins enclosed within phospholipid bilayer vesicles. Various reactions will be cause to occur within these vesicles and changes in size and shape will be measured by, light or electron microscopy, and by conventional and quasielastic light scattering.
