9316025 Newman The effect of the state of G-actin on its interactions, polymerization kinetics and polymer structure will be investigated by a combination of dynamic light scatting, rheology, and fluorescence microscopy techniques. Dynamic light scattering and rheology experiments will be performed to determine the effects of the solvent (divalent cation, nucleotide, and temperature) and the presence of certain actin binding moieties (including the two light chain isoforms of myosin S-1, profilin, and cytochalasin D) on the kinetics and state of actin self-association. Further measurements in the presence of various high concentrations of an inert low molecular weight polymer (polyethylene glycol, PEG) will study the effects of crowding on the kinetics, bundling, and steady-state sizes and dynamic properties of actin filaments. The self- association of actin under crowded conditions, using high concentrations of sucrose or PEG, within artificially-prepared vesicles and within certain epithelial cell types will also be studied using fluorescence imaging microscopy techniques. These studies will provide detailed information on the self-interactions of G-actin and the dynamic properties of the oligomers, filaments, and actin bundles, both in vitro and in vivo, as functions of the solvent and actin-specific binding molecules using a variety of experimental techniques. %%% Three different physical techniques will be used to study the structure and interactions of the protein actin, one of the most common proteins found in nature. Actin's role in determining the structure and mechanical properties of cells is incompletely known. It is clear, however, that actin plays a unique role in generating forces within a cell in order to change the cell's shape or to allow the cell to move actively, as many cells can. A variety of other proteins have been discovered which interact specifically with actin and either regulate or modify the association of many actins togethe r to form long filaments which are capable to sustaining a force. The proposed research will, in part, attempt to elucidate the role of several of the more prominent such proteins by studying their effects on actin in a test tube, in an "artificial" cell constructed from lipid vesicles with actin incorporated inside, and in a living cell. Methods used will include dynamic laser light scattering, viscoelasticity measurements, and fluorescence microscopy techniques. Two main areas of experiments are planned. The first will probe the effects of specific proteins, salts, or drugs on the self- association of actins. Such experiments will help us to understand the details of filament formation in cells leading to eventual force generation, and some of the control mechanisms involved. The second area will study the effects of crowded conditions, such as are found within cells, on the actual self-association rates and final states. Most solution experiments are carried out with purified proteins in the presence of only other small molecules. Recently it has been demonstrated that in the presence of other large macromolecules, not only the rates of self-interaction, but also the final aggregation state can be greatly influenced. Crowding will be produced by the addition of inert macromolecules, which do not directly interact with actin, in order to simulate the actual conditions within cells. These experiments will be carried out both in solutions and in artificial cells, and will be compared to experiments performed in living cells using fluorescence methods. In this way we will attempt to study the interactions of well-characterized proteins in much simpler environments that those of cells, but be able to draw meaningful conclusions about the role of these interactions in living cells.
