Synaptophysin has a selective distribution in early endosomes of PC12 cells

We have studied the endocytic pathway in PC12 cells and localized synaptophysin to a subpopulation of early endosomes. Endocytosis was examined by electron microscopy using horseradish peroxidase as an endocytic tracer. Immediately following brief incubations with horseradish peroxidase, label was seen in small vesicles and tubules which appeared to be part of the tubular early endosomal network. A large vacuolar structure, containing a rim of horseradish peroxidase reaction product and an electron-lucent central region, was also labelled at the earliest time point examined. Within 5 min after the horseradish peroxidase pulse, reaction product was seen in multivesicular bodies. After prolonged chase periods, horseradish peroxidase label was lost from the early endosomal structures and accumulated in large dense vesicles containing lamellar stacks of membranes. The observed pattern of horseradish peroxidase distribution is consistent with delivery of the tracer into tubular and vacuolar early endosomes with subsequent movement of horseradish peroxidase out of these compartments and into lysosomes. Examination of synaptophysin distribution by EM immunocytochemistry following incubations with horseradish peroxidase revealed selective immunogold labelling of early endosomal structures. Notably, small vesicular and tubular profiles were frequently double-labelled while vacuolar early endosomes were only rarely labelled for synaptophysin. Immunocytochemical labelling was not observed in multivesicular bodies or large dense vesicles with lamellar stacks. Results of experiments in which endosomal structures were immunoprecipitated with antibody to synaptophysin were consistent with the immunocytochemical findings. Maximal recovery of endocytosed horseradish peroxidase activity was seen immediately following the horseradish peroxidase pulse, and a significant decrease was seen after brief chase periods. These results indicate the presence of synaptophysin in vesicles and tubules of the early endosomal compartment.