Calcium-Sensitive, Actin-Related Gelation of Cell Extracts of Thermosensitive Chinese Hamster Lung Cells Capable of Anchorage-Independent Growth

Abstract

The extent of actin-related gelation of extracts of thermosensitive Chinese hamster lung (CHL) cells capable of anchorage-independent growth was studied quantitatively by monitoring the total protein in the gel obtained by low-speed centrifugation. The gelation depended on the presence of ATP, KC1, MgCl₂, and a reducing agent. Micromolar concentrations of Ca²⁺ and low doses of cytochalasin B inhibited the actin-related gelation of these extracts. The gelation was more sensitive to inhibition by Ca²⁺ and cytochalasin B when the extracts were prepared from cells cultured at the permissive rather than the nonpermissive temperature. When various ts mutants were examined, the half-maximal inhibitory dose (HMID) of Ca²⁺ and cytochalasin B for gelation of extracts of cells cultured at the nonpermissive temperature was between 1.25 and 2.19 times higher than that for extracts of the same cells cultured at the permissive temperature. The values of the HMID for Ca²⁺ and cytochalasin B changed shortly after the shift in temperature of cell cultures from the nonpermissive to permissive temperature. When cell extracts were incubated at the permissive temperature in vitro for only 15 minutes, these changes in values of HMID were also observed. Analysis of polypeptides of cell extracts and gel pellets on polyacrylamide gel electrophoresis suggested that the decrease in amount of a high-molecular-weight actin-binding protein (250 kDa) may play an essential role in the increased sensitivity to inhibition by Ca²⁺ and cytochalasin B of actin-related gelation in extracts of these ts mutants.