Intraepidermal cell surface fine structure: preservation and examination at high resolution

Abstract

Morphological and functional studies on cell surfaces have been limited largely to cultured cells because of injury wrought to cells of solid tissues by commonly employed mechanical, enzymatic, or chelator dispersal methods. By using the staphylococcal epidermolytic toxin we avoided this problem; the toxin cleaves the intercellular spaces of human and mouse squamous epithelia without ultrastructural evidence of cytotoxicity. We studied the cell surface topography of neonatal mouse epidermis obtained two hours after injection of highly purified epidermolytic toxin. Immediately after sacrifice intraepithelial surfaces were exposed while the animals were immersed in fixative. Specimens were either freeze-fractured or embedded for transmission electron microscopy, or were critical-point-dried prior to platinum/carbon replication for transmission and scanning electron microscopy. Replicas could be prepared for transmission electron microscopy only if they were first stabilized with parloidion and then cleaned with both bleach and 40% chromate. By using these four complementary morphological methods (freeze-fracture, scanning electron microscopy, transmission electron microscopy of surface replicas, and standard thin sections), we could positively identify external membrane structures. The convoluted surface was studded by tenuous microvilli, scattered 15-20 nm particles, and hemispherical desmosomal mounds. Desmosomal plaques displayed randomly arrayed 15-20 nm globular particles comparable in distribution and density to particles observed in freeze-fractured desmosomes, and suggesting that desmosomal integral membrane particles span the external leaflet of the plasma membrane.