Actin-associated proteins related to focal and close cell-substrate contacts in murine fibroblasts

Abstract

Based on the premise that cell-surface proteins involved in focal or close cell-to-substrate contact will be linked across the membrane, either directly or indirectly to F-actin of the cytoskeleton, we have used a modification of the myosin affinity technique (Leonardi, C L & Rubin, R W, Anal biochem 118 (1981) 58) [20] to precipitate F-actin filaments and associated proteins from Nonidet P-40 solubilized cells. BALB/c 3T3 fibroblasts, which have close and focal contacts, and their adhesion-defective mutant, AD6, which has only close contacts were compared. Clarified cell homogenates were incubated with rabbit muscle myosin thick filaments and were pelleted. Supernatants obtained from successive washes of the pellets, before and after release of actin and associated proteins by adenosine triphosphate, were analysed by gel electrophoresis under reducing conditions. The metabolic origin of the actin-associated proteins was determined by [ 35S]methionine labelling of BALB/c 3T3 cells. By these methods, 38 and 57 kD proteins were found to be actin-associated in BALB/c 3T3 but not in AD6. Biochemically reverting AD6 to the BALB/c 3T3 phenotype restored focal contacts and the association of the 38 and 57 kD proteins to actin, further implying roles for the proteins in the focal contact. Proteins of 48 and 145 kD were actin-associated in all three cell types indicating a potential role for these proteins in the close contact.