lucifer-yellow and 3-methyladenine

The intracellular parasite Leishmania survives and proliferates in host macrophages. In this study we show that parasitophorous vacuoles of L. mexicana gain access to cytosolic material via two different routes. (1) Small anionic molecules such as Lucifer Yellow are rapidly transported into the vacuoles by an active transport mechanism that is sensitive to inhibitors of the host cell's organic anion transporter. (2) Larger molecules such as fluorescent dextrans introduced into the host cell cytosol are also delivered to parasitophorous vacuoles. This transport is slower and sensitive to modulators of autophagy. Infected macrophages were examined by two novel assays to visualize and quantify this process. Immunoelectron microscopy of cells loaded with digoxigenin-dextran revealed label in multivesicular endosomes, which appeared to fuse with parasitophorous vacuoles. The inner membranes of the multivesicular vesicles label strongly with antibodies against lysobisphosphatidic acid, suggesting that they represent a point of confluence between the endosomal and autophagosomal pathways. Although the rate of autophagous transfer was comparable in infected and uninfected cells, infected cells retained hydrolyzed cysteine proteinase substrate to a greater degree. These data suggest that L. mexicana-containing vacuoles have access to potential nutrients in the host cell cytosol via at least two independent mechanisms.

Topics: Adenine; Animals; Autophagy; Biological Transport, Active; Cysteine Endopeptidases; Cytosol; Dextrans; Female; In Vitro Techniques; Isoquinolines; Leishmania mexicana; Macromolecular Substances; Macrophages; Mice; Mice, Inbred BALB C; Microscopy, Immunoelectron; Vacuoles

Pathogenic strains of Helicobacter pylori cause progressive vacuolation and death of epithelial cells. To identify the nature of vacuoles, the distribution of markers of various membrane traffic compartments was studied. Vacuoles derive from the endocytic pathway since they include the fluid-phase marker Lucifer yellow. Early endosome markers such as rab5, transferrin, and transferrin receptor, as well as the lysosomal hydrolase cathepsin D, are excluded from these structures. In contrast, the vacuolar membrane is specifically stained by affinity-purified antibodies against rab7, a small GTPase, localized to late endosomal compartments. The labeling of rab7 on vacuolar membranes increases as vacuolation progresses, without a concomitant increase of cellular rab7. Cell vacuolation is inhibited by the microtubule-depolymerizing agents nocodazole and colchicine. Taken together, these findings indicate that the vacuoles specifically originate from late endosomal compartments.

Topics: Adenine; Biomarkers; Cathepsin D; Colchicine; Fluorescent Antibody Technique; GTP-Binding Proteins; HeLa Cells; Helicobacter pylori; Humans; Immunoblotting; Isoquinolines; Kinetics; Nocodazole; Receptors, Transferrin; Vacuoles