Acidification of macrophage and fibroblast endocytic vesicles in vitro.

Abstract

We have used the pH-dependent fluorochrome fluorescein-dextran (FD) to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured macrophages and fibroblasts. FD was internalized by pinocytosis under conditions that allowed its selective localization in endosomes (1-to 5-min pulse) or in lysosomes (5-min pulse, 30-min chase). Fibroblasts were also exposed to FD at 20°C, at which temperature endosome-lysosome fusion is inhibited. Cells were homogenized and labeled organelles were separated by centrifugation in Percoll density gradients. The addition of ATP rapidly decreased the internal pH of both endosomes and lysosomes, as indicated by a decrease in fluorescence intensity. The pH gradient was dissipated by H+ ionophores and ammonium.chloride. Acidification was not affected by inhibitors of the mitochondrial F1,F0-ATPase or the Na+, K, K+-ATPase and did not require permeant anions, Na+, or KV. Of the inhibitors tested, only N-ethylmaleimide prevented the ATP-dependent acidification of both compartments. These findings provide direct support for the existence of an acidic prelysosomal compartment that may be acidified via the same type of H+ pump believed to operate in lysosomes and.secretory granules.Pinocytosis typically results in the accumulation and degradation of internalized macromolecules in secondary lysosomes (1). However, delivery to lysosomes requires the participation of at least two other classes of endocytic vacuoles. Pinocytic vesicles, often with clathrin-containing coats, form at the plasma membrane and constitute the primary endocytic compartment. These vesicles subsequently fuse with, or fuse together to form, a somewhat larger class of secondary endocytic vacuoles (0.2-1.0 ,um in diameter), referred to here as endosomes (1,2). Endosomes can be distinguished from lysosomes in several ways: (i) they have a relatively low buoyant density (3-8); (ii) they are labeled by pinocytic markers more rapidly than and prior to labeling of lysosomes (7,9); (iii) internalized macromolecules exhibit only a transient residence in endosomes, whereas lysosomes are typically the final destination (9); and (iv) endosomes are relatively devoid of acid hydrolases (3,(6)(7)(8).These differences notwithstanding, endosomes may be similar to secondary lysosomes in at least one significant characteristic, low internal pH. This has been suggested by recent studies in which receptor-bound ligands were coupled to fluorescein (whose fluorescence spectrum is a titratable function of pH) and found to be internalized into an acidic compartment prior to delivery to lysosomes (8,10). This was the case also with Semliki Forest virus (SFV), which requires a pH of <6 for fusion and penetrates into the cytosol from prelysosomal vacuoles (11). Here, we have used isolated endosomes labeled with the pH-sensitive fluorochrome fluorescein-dextran (FD) (12, 13) to study more directly the acidification of prelysosomal vacuoles. Two points are made: (i) both isolated endosomes and lysosomes c...

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

“…Depletion of VHA-8 blocks V-ATPase activity, whose function is crucial for maintenance of organelle acidity 30 . Th us, perturbation of V-ATPases leads to organelles showing altered acidities 31,32 . Accordingly, at 5 and 17 min, the vesicles appear highly acidic ( Supplementary Fig.…”

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly

“…FITC-Dextran added to the medium of cells is taken up by fluid-phase endocytosis and is trafficked through the endocytic pathway to lysosomes, where it accumulates (16,17). Following the loading period with a chase of at least 30 min ensures that FITC-Dextran is localized only to lysosomes (16). Because FITC fluorescence is quenched at low pH, the fluorescence signal permits the measurement of lysosomal acidification.…”

“…The following day, the medium was replaced with medium containing 2.2 mg/ml FITC-Dextran, and cells were incubated overnight to allow uptake of the dye by endocytosis. The following day, FITCDextran-containing medium was replaced with unlabeled medium, and cells were incubated to allow all dye to progress to the lysosomal compartment (16). Cells were treated with amino acids and inhibitors as described above.…”

Alterations of intracellular pH homeostasis in apoptosis: origins and roles

“…Although nuclear pH was not measured in their studies, there was evidence suggesting that nuclear pH is lower than cytosolic pH, due to the activity of a nuclear H þ -ATPase. 35 This protein, which appears to be identical to the proton pump mediating a 2 pH unit gradient across the vacuolar membrane, 36 might induce a large acidification in the nucleus upon even limited changes of extranuclear pH. The fact that DNase II is normally dormant and is a ubiquitous enzyme in mammalian tissues implies that its role in apoptosis-related DNA degradation would be important, especially in cells containing an important proportion of such a DNase over others and upon acidotic/apoptotic stimuli.…”