Figure 4.

Fusion of GPCs with PM by correlative light-EM. A VSVG–GFP transfected cell (a–c) was fixed exactly when one of the Golgi-derived fluorescent spots (a and b, arrowheads) started to disappear (b, arrowhead; movie 4.2). The cell surface was then labeled without permeabilization using antibodies against the VSVG ectodomain detected by anti–rabbit Cy3-conjugated antibodies (c, red). Both fuzzy- (c, arrowhead) and sharp-appearing (c, arrow) GPCs were accessible by antibodies, suggesting their contact with the PM. The same approach was used to stain another GPC undergoing fusion (movie 6.3; d–g, arrowhead) using HRP-conjugated secondary antibodies. After resin embedding (f) and sectioning (g), the GPC (f and g, arrowhead) could be easily located using the microvilli (e–g, arrows) as reference marks. Double-labeling of VSVG using immunoperoxidase and immunogold protocols shows patches of both HRP labeling and 10-nm gold particles (h, small arrows) at the GPC fusion site (h, arrow). Bar: (a–c) 9.5 μm; (d–f) 7.5 μm; (g) 930 nm; and (h) 480 nm.