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Fig. 4. The RFP-KDEL protein was not localized on the reticular ER, but was only seen in the vesicular sub-compartment of ER. (A) Expression of RFP-KDEL in a hippocampal neuron observed with a CCD camera. Arrowheads indicate branch points of the dendrites. (B) Double-labeling with RFP-KDEL and GFP-KDEL. In contrast to GFP-KDEL that labeled both reticular ER and vesicles, RFP-KDEL fluorescence was found only on vesicles. RFP-KDEL and GFP-KDEL co-localized on the same vesicles (arrowheads). (C) Representative movements of vesicles labeled with RFP-KDEL. The net movement of each vesicle (µm) was plotted against time (seconds). (D) Double-labeling with RFP-KDEL and endogenous Ins(1,4,5)P₃R1 using monoclonal antibody 18A10. Endogenous Ins(1,4,5)P₃R1 was also found on the RFP-KDEL-labeled vesicles (arrowheads). (E,F) Double-labeling with RFP-KDEL (red) and fluorescein-dextran (E; green), or MitoTracker Green FM (F; green). The upper images are high power micrographs of the soma and the lower images are those of the dendrites. The lysosomal and mitochondrial signals that appear to be outside the transfected cells arise form surrounding cells. RFP-KDEL did not co-localize with these markers. B,D-F are confocal micrographs. Scale bars: 10 µm.

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