Exchange Kinetics and Composition of Endocytic Membranes in Terms of Plasma Membrane Constituents: a Morphometric Study in Macrophages

Intracellular membrane traffic, during endocytosis in mouse bone marrow-derived macrophages, was studied quantitatively by morphometric and kinetic analysis. Three functionally different markers were used: Horseradish peroxidase (HRP) served as a fluid-phase (FP) marker (1000 micrograms HRP/ml in the presence of mannan) or as a receptor-mediated (RM) membrane marker (25 micrograms HRP/ml) and, third, plasma membrane (PM) glycoconjugates, enzymatically labeled with [3H]galactose at the cell surface, served as a covalent membrane marker. The cell surface was labeled with [3H]galactose, followed by either FP or by RM uptake of HRP. The kinetics of the intracellular appearance of the markers were measured as the membrane area stained by HRP-reaction product and as the number of autoradiographic grains associated with these membranes. The following compartments were distinguished: PM, coated vesicles (VI), pinosomes or endosomes (VII), secondary lysosomes (VIII), and HRP-negative vesicles (EV). Tubular structures of VII became labeled with HRP only during RM uptake. The markers flowed first into VI and VII, and after 5 min into VIII. EV became labeled with the covalent membrane marker starting from 5 min. The ratio of autoradiographic grain number to HRP-stained membrane area remained constant with time although substantially different for the various compartments, viz. 100% (VI), 50% (VII and EV) and 30% (VIII) as compared to the PM (100%). This indicated that endosomes were only partially derived from internalized PM and that secondary lysosomes contained a substantial pool of PM constituents. The observed kinetics suggested that once every 30 to 40 min the entire PM was internalized, the bulk of which was recycled after 4 min from a prelysosomal compartment(s) leaving only 12 to 20% for recycling via membranes of secondary lysosomes after a residence time of 24 to 33 min.