Figure 4.

Astrocytes transfected with REST/DBD-GFP express DCVs containing Sg2 and NPY. (A and B) Representative confocal images of GFP and Sg2 compared in two transfected astrocytes. The control astrocyte (A) transfected with GFP remains negative for Sg2; the astrocyte transfected with the REST/DBD-GFP construct (B) also expresses the cargo protein concentrated in small puncta distributed preferentially in the perinuclear area and aligned in the proximity of the plasma membrane. (C) Most Sg2-positive puncta of REST/DBD-GFP–transfected astrocytes are also positive for the endogenous secretory peptide NPY. (D and D’) The conventional ultrastructure of DCVs expressed by the REST/DBD-GFP–transfected astrocytes. (E) Three DCVs, one deep in the cytoplasm, one adjacent to the surface, and one in the process of being exocytized, all strongly immunogold labeled for Sg2. (F–I) Astrocytes, all transfected with REST/DBD-GFP (see their GFP fluorescence in the insets) in which the distribution of Sg2 puncta does coincide only to a background level with those of the markers of TGN (TGN38; F), lysosomes (Lamp1; G), early endosomes (EEA1; H), and late endosomes (TfR; I). The images shown are representative of results obtained in at least three experiments. Bars: (A and B) 20 µm; (C) 5 µm; (D) 500 nm; (D’ and E) 200 nm; (F–I) 20 µm.