Phase equilibria of cytoplasmic actin of cultured epithelial (BHK) cells

The actin in cold lysates of cultured baby hamster kidney cells was predominantly monomeric, as judged by the DNase inhibition assay. Most of the filamentous actin attached to the membranes could be dissociated by washing with low ionic strength buffer, but a minor part was tightly associated in complexes with other proteins and could not be liberated for assay by DNase after exposure to a depolymerizing medium. The monomeric actin in the cell extract must be sequestered as a complex with a profilin-like protein, since its concentration was greatly in excess of the critical concentration for polymerization. On warming, rapid polymerization ensued, with loss of most of the monomeric actin. A separate cross-linking process could be observed by light-scattering measurements. A gel was formed, that contained besides actin substantial amounts of constituents identified as filamin, myosin, a-actinin, a gelsolin-like protein and other species of apparent subunit molecular weights 58 000 and 300 000, presumed to derive from intermediate filaments. Some low molecular weight protein subunits, including tropomyosin, were also present. The gelation was not accompanied by a significant degree of phosphorylation of any of the actin-associated proteins, with the exception of the minor species of 300000 molecular weight, the phosphorylation of which was cAMP-dependent. In the presence of concentrations of free calcium in the sub-micromolar range, the gel initially formed contracted and gradually dispersed. At higher concentrations of free calcium a loose turbid gel formed, but the bulk of the actin was found in the supernatant after high-speed centrifugation. Using a fluorescent actin derivative as a tracer, the quantum yield of which increases by nearly two orders of magnitude when it is incorporated into a polymer, it was established that the actin in the supernatant was not monomeric, but rather in the form of very short filaments. It was shown that scission of filaments accompanied the contraction of the cytoplasmic gel at the lower concentrations of calcium. The short filaments were highly effective in nucleating the polymerization of added G-actin and were evidently rapidly dissociated by DNase, since they reacted as monomers in the DNase inhibition assay. The gel obtained in the absence of calcium was revealed by electron microscopy to consist of clusters of filaments, many of which show a highly regular lateral spacing of about 7·0 nm. The physical basis for the t