| A new actin modifying protein which crosslinks and bundles actin filaments has been identified. Exogenously added calf gamma crystallin, a family of 20KD monomeric lens proteins, modifies the rate, extent and morphology of rabbit skeletal muscle actin polymerization in vitro. It is proposed that gamma crystallin contains two binding sites for actin, at gamma crystallins' lysine residues. The experimental results which establish this finding are: (1) The production of an altered network of polymerized actin filaments: Ultrastructural examination of polymerized actin filaments revealed an aggregated network of intertwined and twisted filaments which formed closely associated bundles. (2) A decreased critical concentration of actin: The polymerization of actin, monitored by the fluorescent probe 1-N-pyrene-iodoacetamide covalently linked to cysteine 374 of actin, demonstrated a lower critical concentration, i.e., the minimum concentration of actin monomer required to produce polymerization. (3) An increased apparent viscosity: The low shear viscosity of polymerized actin filaments as monitored by falling ball viscometry was increased in the presence of gamma crystallin. (4) An altered high shear viscosity: The addition of gamma crystallin to polymerizing actin filaments produced altered apparent rates of polymerization altered final specific viscosities. (5) Carbamylation of lysine residues: The epsilon-amino groups of the two lysine of the gamma crystallins were carbamylated by addition of methylisocyanate. (a) An ultrastructural examination of in vitro actin filaments revealed a loss of bundling and crosslinking properties of gamma crystallin following lysine carbamylation. (b) High shear viscosity measurements showed no increase in the final specific viscosity of polymerized actin filaments with carbamylated gamma crystallin.; The appearance of actin filaments produced by the addition of gamma crystallin, or polygonal arrays, directly preceded stress fiber formation of actin filament bundles in the direction of cell elongation. The synthesis of gamma crystallin is concurrent with the differentiation and elongation of the lens fiber cell. Gamma crystallin could act as a cytoskeletal organizer and may be the necessary component in vivo to produce the stress fibers which are essential for the proper development and morphology of the lens fiber cell. (Abstract shortened with permission of author.)... |
