Ca2+-sensitive isolation of a cortical actin matrix fromDictyostelium amoebae

Abstract

A cortical actin matrix has been isolated from amoebae ofDictyostelium discoideum grown in liquid culture. The existence of this actin matrix in whole cells is indicated in electron micrographs as an area free of cytoplasmic organelles. The actin beneath the membrane is more clearly visible in sections of cells that are lysed gently with 0.5% Triton X-100 and fixed with 1% glutaraldehyde. Such Triton-lysed cells have fragments of plasma membrane associated with the cortical actin matrix. Isolation of the actin matrix, which sediments at 400g, is inhibited by Ca²⁺. As much as 50% of the actin of the cell and about 12% of the total protein is found in the matrix isolated in lysis buffer containing no added Ca²⁺ and 2.5mm EGTA, whereas less than 15% of the actin of the cell is recovered in a 400g pellet when cells are lysed in buffer containing 2.5mm Ca²⁺ and 2.5mm EGTA. A 40 000 molecular weight protein that fragments F-actin in a Ca²⁺-dependent manner is not found in the isolated cortical actin matrix.