Tubular lysosomes and their drug reactivity in cultured resident macrophages and in cell-free medium

Abstract

Lysosomes were assessed in normal living resident mouse peritoneal macrophages, using mainly phasecontrast microscopy (PCM), darkfield microscopy (DFM), and fluorescence microscopy (FM) after terminal acridine orange (AO) staining; these procedures avoided dyes during experimentation. After a few hours of culture a variable proportion of the normal spherical lysosomes began to assemble in a linear fashion. Fully formed tubular structures, with appearances generally recognized as characteristic of tubular lysosomes (TL), could be seen by PCM or, after labeling, by FM, at 2–5 days (best usually at 4–5 days). This peak was followed by a reduction, and at 8–10 days most of the TL had disappeared, leaving only spherical lysosomes. Renewal of the medium at this stage was followed by a temporary reappearance of TL, suggesting that the medium was a major factor in their initial development also. Formation of TL was enhanced by chloroquine (Cq), though to a lesser degree than by phorbol ester (PMA); in contrast NH 4Cl (like Cq a weakly basic amine) caused their disassembly into spherical lysosomes. Manual disruption of the monolayer macrophages enabled TL to be transferred to a cell-free medium, in which they remained apparently stable for several hours. Two known microtubule depolymerizers caused disassembly of TL in the intact cells, reinforcing the idea that the TL are associated with the cytoplasmic microtubule (MT) system; but these agents were inactive in vitro, suggesting that disorganization of the system was responsible for this change. With AO, and more precisely by darkfield microscopy (DFM) of the untreated isolated TL, apparently discrete vesicles could be seen within the tubes, often closely packed, and recalling those reported by others in chemically fixed preparations, but which were then suspected to be fixation artifacts. After considering an alternative interpretation involving single enlarged lysosomes, we interpret our observations as indicating that two or more normal lysosomes assemble to form vesicle-containing tubular structures (which may become stretched along cytoplasmic MT), and that normal lysosomes are restored to the cytoplasm after disassembly. The origin of the tubular boundary membrane is not yet clear.