Substrate-attached membranes of cultured cells isolation and characterization of ventral cell membranes and the associated cytoskeleton

Abstract

We describe here an approach for the isolation and characterization of substrate-attached membranes of cultured cells. The procedure for ventral membrane preparation is based on a short incubation with ZnCl 2, followed by shearing with a stream of buffer. By varying the intensity of shearing it was possible to obtain reproducibly either entire ventral membranes or highly enriched focal contacts. The contacts with the substrate were retained in these preparations in an apparently intact state as determined by interference-reflection microscopy as well as by scanning and transmission electron microscopy. The formation of close contacts by the cells and by the isolated membranes was sensitive to changes of pH value. Thus in buffers at pH 7.0 to 7.2 the attachment was mediated predominantly by focal contacts, whereas at pH 6.0 the membranes reversibly formed extensive close contacts with substrate. The mechanical shearing removed most of the cytoskeleton, leaving attached only those components which were most tightly associated with the ventral membranes. Microtubules were easily removed, together with most of the intermediate filaments, whereas a considerable portion of the microfilament system was retained even after extensive shearing. Immunofluorescent labeling with antibodies to several microfilament-associated proteins, including actin, vinculin, α-actinin, filamin and tropomyosin, pointed to the specific interaction of each of these proteins with the isolated ventral membranes and focal contacts.