Plasma membrane plasticity of Xenopus laevis oocyte imaged with atomic force microscopy

Proteins are known to form functional clusters in plasma membranes. In orde r to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of nativ e plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L- lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first ident ified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Mem brane patches exhibiting the typical phospholipid bilayer height of 5 nm sh owed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently foun d membrane protein structure (40/ mu m(2)) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated gl ass surface while the lipid bilayer was found highly mobile. We detected pr otein structures with distinguishable subunits of still unknown identity. S ince X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level. Copy right (C) 2000 S. Karger AG, Basel.