Figure 6.

Ultrastructure of VSVG–GFP and VSVG containing GPCs. Cells transfected with VSVG–GFP (a and b) or infected with ts-045 VSV (c–g) were fixed and prepared for immunoelectron microscopy using antibodies against the lumenal domain of VSVG. During the 20°C block, VSVG–GFP accumulated in the TGN and in the medial and trans-cisternae of the Golgi stack, whereas the cis compartment remained unstained (a, arrows). Upon warm-up from 20 to 32°C, VSVG–GFP labeling was found not only in the trans-most cisternae of the Golgi (b, arrowheads) and the TGN, but also in tubular structures (b, arrows) detached from the Golgi stack. The PM was also labeled. The same type of labeled tubular structures (arrows) positioned near the PM or Golgi stack were also visible in the cells shifted for 50 min from 40 to 32°C (c) without a 20°C block. Peripheral GPCs appeared both as elongated (d, arrows) or saccular structures (d, arrowhead). To check for colocalization with the endocytic structures, cells were labeled with anti-VSVG antibodies after uptake of WGA-gold conjugate as an endocytosis marker (e). It is evident that no endocytic tracer was found within the GPC (e, arrow), whereas the PM (e, empty arrowheads) and multivesicular body (e, filled arrowheads) were labeled with WGA-gold. Some VSVG-positive tubules (f, arrow) appeared to be connected to the PM. In some cases, an intensely labeled area appeared on the cell surface (g, arrow) presumably in relation to GPC fusion sites. Bar: (a, b, d, e, and g) 500 nm; and (c and f) 400 nm.