Heterogeneity in ATP-dependent acidification in endocytic vesicles from kidney proximal tubule. Measurement of pH in individual endocytic vesicles in a cell-free system.

Measurement of membrane transport in suspensions of isolated membrane vesicles provides averaged information over a potentially very heterogeneous vesicle population. To examine the regulatory mechanisms for ATP-dependent acidification, methodology was developed to measure pH in individual endocytic vesicles. Endocytic vesicles from proximal tubule apical membrane of rat kidney were labeled in vivo by intravenous infusion of FITC-dextran (9 kD); a microsomal fraction was obtained from dissected renal cortex by homogenization and differential centrifugation. Vesicles were immobilized on a polylysine coated coverglass and imaged at high magnification by a silicon intensified target camera. ATP-dependent acidification was not influenced by endosome immobilization. Endosome pH was determined from the integrated fluorescence intensity of individual labeled vesicles after background subtraction. Calibration studies with high K and nigericin showed nearly identical fluorescence vs. pH curves for different endosomes with a standard deviation for a single pH measurement in a single endosome of approximately 0.2 pH units. In response to addition of 1 mM MgATP in the presence of K and valinomycin, endosome pH decreased from 7.2 to a mean of 6.4 with a unimodal distribution with width at half-maximum of approximately 1 pH unit. The drop in endosome pH increased and the shape of the distribution changed when the time between FITC-dextran infusion and kidney removal was increased from 5 to 20 min. Differences in ATP-dependent acidification could not be attributed to heterogeneity in passive proton conductance. These results establish a direct method to measure pH in single endocytic vesicles and demonstrate remarkable heterogeneity in ATP-dependent acidification which was interpreted in terms of heterogeneity in the number and/or activity of proton pumps at serial stages of endocytosis.     

CLIP-170 links endocytic vesicles to microtubules.

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BIK1 proteins. These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.     

Separation of endocytic vesicles in Nycodenz gradients

The endocytosis of 125I-labeled asialofetuin by rat hepatocytes was studied using Nycodenz/sucrose gradients. It was shown in pulse chase experiments that the ligand endocytosed initially (after 1/2 to 1 min) was in small, slow-sedimenting vesicles of similar sizes. The vesicles containing the ligand increased in size, and after about 2.5 min 20-30% of the ligand was recovered in larger, faster-sedimenting vesicles. After 15 min almost all internalized ligand was recovered in the fast-sedimenting vesicles. The initial, small endocytic vesicles and the later, larger endocytic vesicles have similar buoyant densities; the maturation of the endosomes can only be revealed by rate sedimentation, not by isopycnic centrifugation. Dissociation of ligand from receptor was found to occur in the larger, faster-sedimenting vesicles. The presence of ammonia inhibited the increase in size of the ligand-containing endosomes. The methods employed here offer the possibility of obtaining endocytic vesicles at various stage of their development for further studies.     

Separation of two populations of endocytic vesicles involved in receptor-ligand sorting in rat hepatocytes

Rat hepatocytes incubated in high K+ buffer (all Na+ is replaced by K+) internalize glycoproteins bearing terminal galactose moieties but are not able to deliver them to lysosomes (Baenziger, J. U., and Fiete, D. (1982) J. Biol. Chem. 257, 6007-6009). Instead, internalized ligand accumulates in a prelysosomal compartment(s) with a density similar to that of plasma membrane. We have separated two populations of prelysosomal endocytic vesicles from hepatocytes incubated in high K+ buffer. The vesicle population VR.L has a mean density of 1.14 by sucrose gradient centrifugation and contains functionally active Gal/GalNAc-specific receptor which is able to bind intravesicular ligand. The vesicle population VL has a mean density of 1.19. It contains ligand, but is deficient in Gal/GalNAc-specific receptor when compared to VR.L. These two vesicle populations appear to arise from intracellular organelles which participate in receptor-ligand segregation in rat hepatocytes. Pulse-chase experiments indicate that ligand passes from VR.L to VL. VR.L and VL are also detected in hepatocytes incubated in buffers containing physiologic amounts of Na+; however, the proportion of ligand found in VL is less than in cells incubated in K+-containing buffer. The primary effect of high K+ buffer is to prevent exit of ligand from VL whereas the accumulation of ligand in VR.L is likely secondary to the effect on VL. Membrane protein constituents of VR.L and VL were identified by vectorial lactoperoxidase labeling using a galactosyl conjugate of lactoperoxidase. Vesicles containing Gal-lactoperoxidase were isolated and labeling initiated by addition of 125I, glucose, and glucose oxidase. The labeling patterns for VR.L and VL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinct from the more complex labeling pattern obtained at the cell surface. Analysis by two-dimensional electrophoresis demonstrated a highly selective labeling pattern with only a small number of differences between VR.L and VL. This suggests that the major membrane components of the compartments prior to and following receptor-ligand segregation are the same. Thus, receptors may be selectively removed from these membranes during the process of receptor-ligand segregation.     

Isolation of rat liver endocytic vesicles using the proton pump as a marker

ATP-driven acidification visualized by the delta pH indicator acridine orange was used as marker for isolation of endocytic vesicles from rat liver. By differential and Percoll density gradient centrifugation, a vesicle fraction was obtained with an approx. 80-fold enriched H+-pump activity. The preparation contained vesicles that had taken up fluorescein isothiocyanate-labeled dextran or horseradish peroxidase injected into rats in vivo, proving the presence of endosomes. The H+-pump in these vesicles showed: (a) strict preference for ATP; (b) stimulation by Mg2+ and Mn2+, but not by monovalent cations; (c) stimulation by Cl-, I- and Br-; (d) electrogenicity; (e) insensitivity to vanadate, slight inhibition by oligomycin and strong inhibition by N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodimide (DCCD). The vesicles exhibited an ouabain-, oligomycin- and levamisole-resistant ATPase activity, which was slightly stimulated by Cl-, unaffected by vanadate and inhibited by NEM and DCCD. Thus, a simple and efficient high-speed centrifugation method is available for isolation of endocytic vesicles from mammalian liver.     

Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.     

Mobilization of iron from endocytic vesicles. The effects of acidification and reduction

The factors necessary to dissociate iron from transferrin in endocytic vesicles and to mobilize the iron across the vesicle membrane were studied in a preparation of endocytic vesicles markedly enriched in transferrin-transferrin receptor complexes isolated from rabbit reticulocytes. Vesicles were prepared with essentially fully saturated transferrin by incubating the reticulocytes with the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prior to incubation with 59Fe, 125I-transferrin with or without fluorescein isothiocyanate labeling. Initiation of acidification by the addition of ATP was sufficient to achieve dissociation of 59Fe from transferrin with a rate constant of 0.054 +/- 0.06 s-1. Mobilization of 59Fe out of the vesicles required, besides ATP, the addition of a reductant with 1 mM ascorbate, allowing approximately 60% mobilization at 10 min with a rate constant of 0.0038 +/- 0.0006 s-1. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x 10(-9) mol of NADH reduced per min/mg of vesicle protein. Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and monensin. Mobilization, but not dissociation, was inhibited by the permeant Fe(II) chelator alpha,alpha'-dipyridyl. The Fe(III) chelators deferoxamine, diethylenetriaminepentaacetic acid, and apotransferrin did not promote mobilization of dissociated iron in the absence of a reductant. This study establishes the basis for the cellular incorporation of iron through the endocytic pathway in which the endocytic vesicle membrane utilizes, in a sequential way, an acidification system, an iron reduction system, and an Fe(II) transporter system.     

Isolation of functional, coated, endocytic vesicles

Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.     

Ca2+ accumulation and loss by aberrant endocytic vesicles in sickle erythrocytes.

Sickle cells contain internal vesicles which accumulate Ca2+. As shown here, the membrane enclosing the vesicles contains the plasma membrane Ca(2+)-ATPase, or Ca2+ pump, as judged by staining with an antibody directed against the protein. Moreover, the number of cells containing such vesicles increases upon deoxygenation. These findings argue strongly that the vesicles arise by endocytosis from the plasma membrane, and explain how they accumulate Ca2+. When sickle cells are depleted of ATP, Ca2+ is lost from the vesicles, as judged by the disappearance of staining with the Ca2+/membrane probe chlortetracycline (CTC), without a corresponding loss of antibody staining. This loss of Ca2+ can be inhibited by nitrendipine, a Ca2+ channel blocker. These results suggest that the vesicle membrane allows outward passage of Ca2+ by a nitrendipine-sensitive pathway, which can be overcome by the inward-directed activity of the Ca2+ pump of the vesicle membrane. If so, the Ca2+ which vesicles contain is in dynamic equilibrium with the cytoplasm of the sickle erythrocyte.     

Prion aggregates transfer through tunneling nanotubes in endocytic vesicles

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrP^Sc. The intercellular transfer of PrP^Sc is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrP^Sc can move between cells. Here we investigate further the details of this mechanism and show that PrP^Sc travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs.     

Prion aggregates transfer through tunneling nanotubes in endocytic vesicles

Abstract

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrP^Sc. The intercellular transfer of PrP^Sc is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrP^Sc can move between cells. Here we investigate further the details of this mechanism and show that PrP^Sc travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs.     

Induction of endocytic vesicles by exogenous C(6)-ceramide.

Ceramide is a newly discovered second messenger that has been shown to cause cell growth arrest and apoptosis. Here, we present evidence that exogenously added C(6)-ceramide induces enlargement of late endosomes and lysosomes. 10 microM C(6)-ceramide caused the formation of numerous vesicles of varying sizes (2-10 micrometers) in fibroblasts (3T3-L1 and 3T3-F442A), without toxic effects. Vesicle formation induced by C(6)-ceramide was time- and dose-dependent, rapid, and reversible. Numerous small vesicles appeared within 8 h of treatment with 10 microM C(6)-ceramide. They enlarged with time, with large vesicles found in the perinuclear region and small ones observed at the cell periphery. Within 24 h of treatment, approximately 30% of the cells exhibited these vesicles. Removal of ceramide from the culture medium caused disappearance of the vesicles, which reappeared upon readdition of ceramide. Confocal immunofluorescence microscopic analysis using an anti-lysosome-associated membrane protein antibody identified the enlarged vesicles as late endosomes/lysosomes. The fluorescent C(6)-NBD-ceramide, a vital stain for the Golgi apparatus, did not stain these vesicles. The effect on vesicle formation was influenced by ceramide structure; D-erythro-C(6)-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as sphingosine, sphingosine 1-phosphate, N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C(2)-ceramide G(M3), (G(M3), N-acetylneuraminosyl-alpha(2, 3)-galactosyl-beta(1,4)-glucosylceramide), were inactive in causing vesicle formation when added exogenously. Together, these studies demonstrate that exogenous C(6)-ceramide induces endocytic vesicle formation and causes enlarged late endosomes and lysosomes in mouse fibroblasts.     

Fractionation of rat ventricular nuclei.

Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.     

Precise positioning of myosin VI on endocytic vesicles in vivo

Myosin VI has been studied in both a monomeric and a dimeric form in vitro. Because the functional characteristics of the motor are dramatically different for these two forms, it is important to understand whether myosin VI heavy chains are brought together on endocytic vesicles. We have used fluorescence anisotropy measurements to detect fluorescence resonance energy transfer between identical fluorophores (homoFRET) resulting from myosin VI heavy chains being brought into close proximity. We observed that, when associated with clathrin-mediated endocytic vesicles, myosin VI heavy chains are precisely positioned to bring their tail domains in close proximity. Our data show that on endocytic vesicles, myosin VI heavy chains are brought together in an orientation that previous in vitro studies have shown causes dimerization of the motor. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function.     

Bi-directional transport of GLUT4 vesicles near the plasma membrane of primary rat adipocytes

Insulin stimulates glucose uptake into adipocytes by mobilizing intracellular membrane vesicles containing GLUT4 proteins to the plasma membrane. Here we applied time-lapse total internal reflection fluorescence microscopy to study moving parameters and characters of exogenously expressed GLUT4 vesicles in basal, insulin and nocodazole treated primary rat adipocytes. Our results showed that microtubules were essential for long-range transport of GLUT4 vesicles but not obligatory for GLUT4 distribution in rat adipocytes. Insulin reduced the mobility of the vesicles, made them tethered/docked to the PM and finally had constitutive exocytosis. Moreover, long-range bi-directional movements of GLUT4 vesicles were visualized for the first time by TIRFM. It is likely that there are interactions between insulin signaling and microtubules, to regulating GLUT4 translocation in rat adipocytes.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin

We have used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F-alpha 2M for 30 min at 37 degrees C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F-alpha 2M for 15 min at 37 degrees C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F-alpha 2M microenvironment was 5.0 +/- 0.2. Using colloidal gold-alpha 2M for electron microscopic localizations we have verified that, under these conditions, alpha 2M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for 1/2 hr, we obtained a pH of 5.0 +/- 0.2 for the F-alpha 2M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 +/- 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.     

Cell-free fusion of endocytic vesicles is regulated by phosphorylation

Okadaic acid and microcystin-LR, both potent inhibitors of protein phosphatases (PP), blocked vesicle fusion in a cell-free system. The effect of okadaic acid was reversed by the purified catalytic subunit of PP2A, but not PP1. Inhibition was gradual, required Mg-ATP, and was reduced by protein kinase inhibitors, indicating that it was mediated via protein phosphorylation. A candidate protein kinase would be cdc2 kinase, which normally is active in mitotic extracts and has been shown to inhibit endocytic vesicle fusion (Tuomikoski, T., M.-A. Felix, M. Dorée, and J. Gruenberg. 1989. Nature (Lond.). 342:942-945). However, it would appear that cdc2 kinase is not responsible for inhibition by okadaic acid. When compared to cytosol prepared from mitotic cells, okadaic acid did not increase cdc2 kinase activity sufficiently to account for the inhibition. In addition, inhibition was maintained when cdc2 protein was depleted from cytosol.     

Acidification of endocytic vesicles by an ATP-dependent proton pump

One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2- macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma- imido)triphosphate (AMP-PNP) could support acidification. The ATP- dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP- dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.