Cell surface events during resealing visualized by scanning-electron microscopy

Abstract

The function of exocytosis during plasma membrane resealing might be to facilitate the flow of surface lipid over the disruption site and/or to add defect-spanning "patches" of internal membrane across it. Scanning-electron-microscopic visualization of large plasma membrane disruptions in sea urchin eggs is here used to distinguish between these two possibilities. Disruptions were induced by shear stress in the presence and absence of resealing-permissive levels of external Ca2+, and the eggs were fixed at various intervals thereafter for microscopic processing. In eggs fixed immediately (<1 s) after shearing in the absence of Ca2+, a condition which prevents resealing, disruption sites were filled with a uniform population of spherical vesicles (approximately 1 microm in diameter). In eggs fixed immediately after shearing at a resealing-permissive level of Ca2+, disruption sites were filled with a highly heterogeneous population of enlarged vesicles, some being more than 10 microm in diameter and many having irregular profiles and/or appearing to be joined to one another. In eggs fixed 2 s or 5 s post-shearing, the continuity of these large vesicles with one another and the surface membrane began to obscure individual vesicle identities. Single "apertures" of discontinuity over disruption sites, the predicted morphology of a flow-based resealing mechanism, were not observed at any time point (1-5 s) during the interval required for completion of resealing. These observations provide strong confirmation that "patching" of large disruptions mediates their resealing.