IMAGING OF THE CYTOPLASMIC LEAFLET OF THE PLASMA-MEMBRANE BY ATOMIC-FORCE MICROSCOPY

The cytoplasmic face of ventral cell membranes of Madin-Darby canine k idney (MDCK) cells grown on glass coverslips was imaged by atomic forc e microscopy (AFM) in air and under aqueous medium, in ''contact'' mod e. Micrometer range scans on air-dried samples revealed a heterogeneou s structure with some filaments, likely corresponding to actin filamen ts that abut the inner leaflet of the membrane, and a few semi-organiz ed lattice structures that might correspond to clathrin lattices. Expe riments in phosphate-buffered saline confirmed the heterogeneity of th e inner membrane surface with the presence of large (> 100 nm) globula r structures emerging from the surface. Using sub-micrometer scan rang es, protruding particles, that occupy most of the membrane surface, we re imaged in liquid medium and in air. These particles, 8 to 40 nm x-y size, were still present following ethanol dehydration which extracts a large fraction of membrane lipids, indicating their proteic nature. Due, at least partly, to the presence of some peripheral proteins, hi gh magnification images of the inner membrane surface were heterogeneo us with regard to particle distribution. These data compare with those previously reported for the external membrane leaflet at the surface of living MDCK cells. They show that details of the cytosolic membrane surface can be resolved by AFM. Finally, the images support the view of a plasma membrane organization where proteins come into close proxi mity.