The mysteries of motion inside living cells must be investigated from both biochemical and biophysical perspectives. The molecular apparatus of cytoplasmic motility includes scores of proteins that have evolved for specialized functions in the complex process of intracellular force generation. Many of these proteins have now been isolated and characterized both chemically and functionally; but the interplay among them to create supramolecular structures and to generate intracellular motive forces is still a matter of more speculation than understanding. Even the most fundamental issues about macromolecular self-assembly and supramolecular hydrodynamics remain to be resolved. The broad objective of Dr. Ware's work in this field is to utilize the techniques of macromolecular physical chemistry to provide quantitation of mechanistically significant observables. The primary component of the motile apparatus is actin, the most abundant and most highly conserved protein in nature. The mechanism of actin self-assembly and its regulations will be investigated using the fluorescence photobleaching recovery technique and other methods including fluorescence spectroscopy, viscosimetry, and light scattering. Specific issues to be addressed include the role of cations in promoting assembly and the role of ATP hydrolysis in determining kinetic and thermodynamics parameters. Particular emphasis will be placed on the determination of filament length and filament number using multiple physical techniques, including fluorescence recovery after photobleaching quasi-elastic light scattering and viscosimetry. Similar experiments will be employed to examine the mechanisms of action of proteins that have regulatory function on actin assembly and filament length. The results of these experiments and related physical investigations on model systems will be interpreted with the goal of providing objective physical tests for proposed mechanisms of regulated actin assembly, cytoplasmic rheology, and cytoplasmic motility.
