The research proposed in this application is directed to the understanding of the mechanisms of formation and the structure of actin networks. Changes in the structure of actin filament networks in the peripheral cytoplasm are involved in a number of processes involving cell motility. These include phagocytosis, cell locomotion, secretion, and cell activation and so are important in such varied processes as the immune response, wound healing, and blood coagulation. Four particular aspects of actin structure are to be studied. 1) The effects of three cytoplasmic actin modulating proteins, gelsolin, ABP/filamin, and myosin on both the kinetics of actin polymerization and the regulation of the balance between monomeric and polymeric actin will be studied using a fluorescent label attached to actin. 2) The structure of the actin modulating proteins and their interactions with actin will be further examined by a variety of optical methods including fluorescence polarization anisotropy and quasielastic light scattering (QLS) which can measure the rates of diffusion of these proteins and so detect changes in their conformation or binding to other proteins. 3) The viscoelastic properties of F-actin solutions and gels will be measured over a range of deformations of the magnitude expected to occur in cells using a specially designed viscoelastometer. These studies will define the physical properties of actin networks and, in combination with various theories, can yield information about the network structure. The effects of other proteins on the viscoelastic properties of actin will be measured and interpreted in light of their biochemical interactions. 4) Both the process of actin polymerization and the steady state structure of F-actin will be examined by QLS and the results compared to complementary measurements by viscoelastic and biochemical techniques.
One aim of these studies will be the development of a QLS technique that can be used to study changes in the structure of actin networks within the cytoplasm of living cells.
