Fusion accessibility of endocytic compartments along the recycling and lysosomal endocytic pathways in intact cells

Abstract

A fluorescence assay developed for the quantification of intracellular fusion of sequentially formed endocytic compartments (Salzman, N.H., and F.R. Maxfield. 1988. J. Cell Biol. 106:1083-1091) has been used to measure the time course of endosome fusion accessibility along the recycling and degradative endocytic pathways. Transferrin (Tf) was used to label the recycling pathway, and alpha2-macroglobulin (α2M) was used to label the lysosomal degradative pathway. Along the degradative pathway, accessibility of vesicles containing α2M to fusion with subsequently formed endocytic vesicles decreased with apparent first order kinetics. The T 1/2 for the loss of fusion accessibility was ~8 min. The behavior of Tf is more complex. Initially the fusion accessibility of Tf decayed rapidly (t 1/2 <3 min), but a constant level of fusion accessibility was then observed for 10 min. This suggests that Tf moves through one fusion accessible endosome rapidly and then enters a second fusion accessible compartment on the recycling pathway. At 18°C, fusion of antifluorescein antibodies (AFA) containing vesicles with F-α2M was observed when the interval between additions was 10 min. However, if the interval was increased to 1 h, no fusion with incoming vesicles was observed. These results identify the site of F-α2M accumulation at 18°C as a prelysosomal late endosome that no longer fuses with newly formed endosomes since no delivery to lysosomes is observed at this temperature.