Fig. 1.

Subcellular targeting of rapsyn–GFP to exocytic compartments in COS-7 cells. Cells were transfected with rapsyn fused to GFP (rapsyn–GFP) as described in Materials and Methods. Twelve to 24 hr after transfection, cells were fixed and handled for immunofluorescence using TRITC-conjugated WGA (C), monoclonal anti-CTR433 antibody (D), monoclonal anti-TGN46 antibody (E, F), polyclonal anti-Rab5a (G), or anti-M6PR antibodies (H). In some experiments, DAPI staining was performed to localize the nuclei. At early expression times, rapsyn–GFP mostly localized in a juxtanuclear region (A). At later expression times, rapsyn–GFP distributed in clusters at the plasma membrane, as well as in the juxtanuclear region, and in a dot-like pattern within the cytoplasm (B). Rapsyn–GFP fluorescence overlapped partially with WGA staining (C) and with thetrans-Golgi network marker TGN46 (E). At higher magnification, confocal analysis confirmed the codistribution of rapsyn–GFP with TGN46 (F). Rapsyn–GFP did not overlap significantly with the markers of CTR433 (D) and the early and late endosomal compartments Rab5a and M6PR, respectively (G, H). Scale bars:A–E, G, H, 10 μm;F, 5 μm.