Figure 7.

Ultrastructural localization of the cytoplasmically expressed GFP-C2 probe; HPF-FS method. BHK cells expressing GFP-Lact-C2. Cells were high-pressure frozen, freeze-substituted, and embedded in UV-polymerized Lowicryl at low temperature. Sections were immunogold-labeled for GFP. (a) Low-magnification overview showing specificity of labeling (top cell transfected, bottom cell untransfected). Note the high labeling on the PM and on multivesicular endosomes (E) and the absence of labeling of the nuclear envelope. (b–f) Gallery of representative images showing labeling of endosomes but low labeling of mitochondria (M) and Golgi cisternae (G). (b) A putative early endosome (EE), recognizable by the bilayered clathrin patch (arrow), shows significant labeling. (c–e) Individual multivesicular endosomes show distinct labeling patterns including cytoplasmic staining (c and d, arrows), predominantly internal labeling (f), a mixture of the two, or unlabeled (c, asterisk). Note the striking labeling of a specific subpopulation of internal membranes in c, d, and the inset (arrowheads). (e and f) Golgi cisternae show negligible labeling, but vesicles and tubules close to the Golgi, putative TGN elements, including clathrin-coated buds (arrows), show significant labeling. Bars, 200 nm.