In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles

Abstract

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca2+-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal.