Characterization of the movement of receptors and ligands through the endocytic apparatus in alveolar macrophages.

A number of studies have outlined general features of the endocytic pathway. Specific details are lacking regarding the factors that regulate the rates of movement of receptors and ligands within the endocytic pathway, as well as the organelles which constitute the pathway. These studies demonstrated that the rates of internalization of different receptor-ligand complexes, in a single cell type, are different, and that the specific internalization rate of a receptor-ligand complex was unaffected by either the degree of receptor occupancy or the simultaneous internalization of different receptor-ligand complexes in alveolar macrophages. Different receptor-ligand complexes were shown to be internalized into the same endosomes. After internalization, the degradation of different ligands was initiated at the same time, suggesting that ligands that move to the degradative compartment move at the same rate regardless of whether ligands were internalized by receptor-mediated or fluid-phase pinocytosis. Different receptors move back to the cell surface at the same rate, regardless of whether they were initially unoccupied or occupied. The fusion of sequentially internalized vesicles was examined in alveolar macrophages and HeLa cells. The results demonstrated that most ligands internalized at different times mixed within early endosomes, thus demonstrating fusion of sequentially internalized vesicles. The fusion of early endosomes appeared to involve a long lived fusion competent vesicle in which receptor and ligand sorting occurred. Transferrin molecules were shown to move into and out of this sorting compartment very rapidly such that sequentially internalized transferrin molecules did not mix in early endosomes. Additionally, sequentially internalized transferrin molecules did not mix in recycling endosomes. Ligands destined for the lysosome appeared to move rapidly into and out of fusion competent early endosomes. Within the late endocytic pathway sequentially internalized vesicles were capable of fusing prior to the lysosome. This later fusion compartment was defined as a late endosome based on the absence of transferrin-transferrin receptors and classic lysosomal markers, such as acid glycosidases. Ligand degradation occurred in this compartment, indicating the presence of proteases. The ability of late endosomes to fuse with demonstrated using lowered temperatures and/or a methodology that inhibited late endosome-lysosome fusion, the substitution of K+ for Na+ in incubation buffers. It was determined that the substitution of K+ for Na+ buffers prevented endosome-lysosome fusion in a number of different cell types. These studies also included an investigation of the mechanisms(s) by which iso-K+ solutions inhibited endosome movement. A number of potential agents or molecules were ruled out as being causal to the inhibition of endosome-lysosome fusion including: pH changes, alteration in microtubules, and increases in intracellular cyclic-AMP or Ca[2+]. Studies using ion substitution, or sucrose addition, identified changes in cell volume as being causal to derangements in late endosome movement.