The assembly of G-actin into F-actin filaments, the cross-linking of those filaments into a supramolecular network, the regulation of the elastic properties of that network, and the transport properties of biological macromolecules within and through that network are fundamental issues related to the motility and viability of all living cells. In this proposal a program is outlined that will apply sophisticated physical techniques, particularly various types of laser quasi-elastic light scattering, to the investigation of specific questions regarding the mechanisms of these fundamental life processes. Electrophoretic light scattering, a technique that was invented and developed by the principal investigator, will be used to study the association of cations with G-actin and the formation of oligomers and short filaments in the early stages of actin assembly. Preliminary experiments indicate that electrophoretically distinct intermediate species may be resolved in real time in the electrophoretic light scattering spectrum. These results will be pursued to identify these species and to quantify the role of electrostatic effects in the assembly mechanism. The same technique will also be used to characterize the actin-profilin complex, including direct measurements by two independent means of its dissociation constant. Quasi-elastic light scattering and viscoelastometry will characterize actin gels formed by cross-linking actin filaments with model cross-linkers and with cytoplasmic regulatory proteins. Elastic moduli and critical gel points will be determined for model cross-linkers as a function of size, electric charge, and concentration ratio in an effort to determine the molecular requirements for efficient cross-linking of actin filaments into networks who physical characteristics are comparable to physiological values. Rheological measurements of cytoplasmic flow both in vitro and in vivo will provide realistic models for transport properties of cytoplasm under physiological conditions in response to chemical and physical stimuli. At the highest level of sophistication the light scattering techniques will be incorporated into an optical microscope for spatial resolution of sol-gel transformations and cytoplasmic transport in living cells.
| Pan, X X; Ware, B R (1988) Actin assembly by lithium ions. Biophys J 53:11-6 |
| Klein, J W; Ware, B R; Barclay, G et al. (1987) Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes. Chem Phys Lipids 43:13-23 |
| Plank, L; Ware, B R (1987) Acanthamoeba profilin binding to fluorescein-labeled actins. Biophys J 51:985-8 |
| Plank, L; Ware, B R (1986) Assembly of Acanthamoeba actin in the presence of Acanthamoeba profilin measured by fluorescence photobleaching recovery. Biochem Biophys Res Commun 140:308-12 |
