Characterization of cross-linked actin filament gels and bundles using birefringence and polarized light scattering

Fundamental processes in the life of Dictyostelium, such as locomotion, endocytosis, cytokinesis, and morphogenesis, are mediated by the actin cytoskeleton, which is composed of actin, myosins, and numerous actin-binding proteins. An understanding of these processes at the molecular level will require characterization of the structure, function, and dynamics of the actin and actin-binding proteins both in vivo and in vitro. Dictyostelium has more than a dozen actin cross-linking proteins that can mediate the formation of isotropic actin gels and/or anisotropic actin bundles. We describe the use of transmitted polarized light and polarized light scattering in studies of actin and actin-binding proteins during formation of nematic, gelled, or bundled structures. These methods have allowed quantitative studies of the effects of actin filament length, the concentration of actin, and the concentration of the cross-linking protein on the formation of cross-linked actin structures. Such methods hold great promise for characterization of novel cross-linking proteins, and for interpretation of phenotypes from strains lacking or expressing altered forms of these proteins. These methods are also applicable to studies of other systems such as the interactions of microtubules and microtubule associated proteins.