Decoration with myosin subfragment-1 disrupts contacts between microfilaments and the cell membrane in isolated Dictyostelium cortices

Abstract

We used isolated cortices from ameboid cells of Dictyostelium discoideum to examine the structural nature of attachments between microfilaments and the cell membrane and to determine the effect of myosin subfragment-1 (S-1) on such contacts. By varying several parameters in our previously described isolation procedure, we have improved this procedure and have been able to isolate stable cortices. In this paper we identify two types of contact sites between microfilaments and the cell membrane similar to those seen in the brush border of intestinal epithelial cells: (a) an end-on attachment between the barbed end of actin filaments and the cell membrane; and (b) a lateral attachment mediated by rod-shaped bridges measuring ~6 x 15 nm. The spacing between bridges averages 36 nm, which suggests that the helical twist of the actin filament influences bridge location. Together these contacts account for an average of ~25,000 attachments per cell. Incubation of cortices with concentrations of S-1 sufficient to saturate binding sites on the microfilaments caused disruption of the contacts. This observation was confirmed by quantitative morphometry to show a threefold loss in the number of contact sites following S-1 decoration. These results indicate that S-1 decoration should be used with caution when information about the precise location of microfilaments and their attachment to the membrane is required.