Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro

Abstract

We have investigated two aspects of membrane traffic at early stages of endocytosis: membrane fusion and microtubule-dependent transport. As a marker, we have used the trans-membrane glycoprotein G of vesicular stomatitis virus implanted into the plasma membrane and then internalized for different times at 37°C. The corresponding endosomal fractions were immunoisolated using the cytoplasmic domain of the G protein as antigen. These fractions were then used in an in vitro assay to quantify the efficiency of fusion between endosomal vesicles. To identify the vesicular partners of the fusion, these in vitro studies were combined with in vivo biochemical and morphological experiments. Internalized molecules were delivered to early endosomal elements, which corresponded to a network of tubular and tubulovesicular structures. Rapid recycling back to the plasma membrane and routing to late stages of the pathway occurred from these early endosomal elements. These elements exhibited a high and specific fusion activity with each other in vitro, suggesting that individual elements of the early endosomal compartment interact with each other in vivo. After their appearance in the early endosome, the molecules destined to be degraded were observed at the next stage of the pathway in distinct spherical vesicles (0.5 μm diam) and then in late endosomes and lysosomes. When the microtubules were depolymerized with nocodazole, endocytosis proceeded as in control cells. However, internalized molecules remained in the spherical vesicles and did not appear in late endosomes or lysosomes. These spherical vesicles had relatively little fusion activity with each other or with early endosomal elements in vitro. Our observations suggest that the spherical vesicles mediate transport between the early endosome and late endosomes and that this process requires intact microtubules.