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.     

Fractionation of endocytic vesicles and glucose-transporter-containing vesicles in rat adipocytes.

We subfractionated intracellular vesicles from rat adipocytes in order to examine the subcellular distribution of endocytic vesicles or endosomes with respect to insulin-regulatable glucose-transporter (GT)-containing vesicles [James, Lederman & Pilch (1987) J. Biol. Chem. 262, 11817-11824]. Vesicles mediating fluid-phase endocytosis sedimented as a single major peak of greater density than the single distinct peak of GT-containing vesicles. This difference was also apparent during cellular insulin exposure and after insulin removal. Endocytosis of insulin and IGF (insulin-like growth factor) II was also examined. In sucrose gradients, IGF II-containing vesicles were less dense than those containing internalized insulin. Receptor-mediated endocytic vesicles were distinct from fluid-phase endocytic vesicles, but overlapped with the GT-containing vesicles. Vesicles containing internalized ligand were further fractionated by agarose-gel electrophoresis after various times of internalization. At least three different vesicle subpopulations containing the iodinated ligands were resolved after 5 min of internalization. Endocytic vesicles containing rapidly internalized insulin (1.5 min at 37 degrees C) consistently co-migrated with GT-containing vesicles. These data indicate that fluid-phase and receptor-mediated endocytosis occur via different pathways in adipocytes. Furthermore, whereas the intracellular GT-containing vesicles are distinct from fluid-phase vesicles, a rapidly labelled pool of insulin-containing vesicles consistently co-fractionated with GT-containing vesicles when separation techniques based on size, density and charge were used. This suggests that the insulin receptor may directly interact with the intracellular GT-containing vesicles after insulin-induced endocytosis.     

Uncoated endocytic vesicles require the unconventional myosin, Myo6, for rapid transport through actin barriers

After clathrin-mediated endocytosis, clathrin removal yields an uncoated vesicle population primed for fusion with the early endosome. Here we present the first characterization of uncoated vesicles and show that myo6, an unconventional myosin, functions to move these vesicles out of actin-rich regions found in epithelial cells. Time-lapse microscopy revealed that myo6-associated uncoated vesicles were motile and exhibited fusion and stretching events before endosome delivery, processes that were dependent on myo6 motor activity. In the absence of myo6 motor activity, uncoated vesicles remained trapped in the actin mesh, where they exhibited Brownian-like motion. Exit from the actin mesh occurred by a slow diffusion-based mechanism, delaying transferrin trafficking to the early endosome. Expression of a myo6 mutant that bound tightly to F-actin produced immobilized vesicles and blocked trafficking. Depolymerization of the actin cytoskeleton rescued this block and specifically accelerated transferrin delivery to the early endosome without affecting earlier steps in endocytosis. Therefore actin is a physical barrier impeding uncoated vesicle trafficking, and myo6 is recruited to move the vesicles through this barrier for fusion with the early endosome.     

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.     

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.     

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.     

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.     

Assessment of myosin II, Va, VI and VIIa loss of function on endocytosis and endocytic vesicle motility in bone marrow-derived dendritic cells

An essential feature of dendritic cell immune surveillance is endocytic sampling of the environment for non-self antigens primarily via macropinocytosis and phagocytosis. The role of several members of the myosin family of actin based molecular motors in dendritic cell endocytosis and endocytic vesicle movement was assessed through analysis of dendritic cells derived from mice with functionally null myosin mutations. These include the dilute (myosin Va), Snell's waltzer (myosin VI) and shaker-1 (myosin VIIa) mouse lines. Non muscle myosin II function was assessed by treatment with the inhibitor, blebbistatin. Flow cytometric analysis of dextran uptake by dendritic cells revealed that macropinocytosis was enhanced in Snell's waltzer dendritic cells while shaker-1 and blebbistatin-treated cells were comparable to controls. Comparison of fluid phase uptake using pH insensitive versus pH sensitive fluorescent dextrans revealed that in dilute cells rates of uptake were normal but endosomal acidification was accelerated. Phagocytosis, as quantified by uptake of E. coli, was normal in dilute while dendritic cells from Snell's waltzer, shaker-1 and blebbistatin treated cells exhibited decreased uptake. Microtubule mediated movements of dextran-or transferrin-tagged endocytic vesicles were significantly faster in dendritic cells lacking myosin Va. Loss of myosin II, VI or VIIa function had no significant effects on rates of endocytic vesicle movement.     

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.     

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.     

Effect of ascorbate in the reduction of transferrin-associated iron in endocytic vesicles

Externally added ascorbate or NADH effectively reduced ferricyanide and promoted the exit of Fe³⁺ originated from acid-destabilized transferrin contained inside endocytic vesicles. The effect of ascorbate was mediated by an ascorbate uptake system, and the effect of NADH was mediated by the membrane-associated oxidoreductase. At physiological concentrations of both ascorbate and NADH, the ascorbate transport and the NADH-oxidoreductase system were additive as measured by the rate of reduction of ferricyanide and by the mobilization of transferrin-associated iron. The results indicate that Fe³⁺ reduction may occur by a nonenzymatic reaction with ascorbate transported into the vesicle lumen. The ascorbate-mediated reduction of iron derived from transferrin occurring in the endosome could play a major role in cellular iron uptake.     

Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro

We have investigated two aspects of membrane traffic at early stages of endocytosis: membrane fusion and microtubule-dependent transport. As a marker, we have used the trans-membrane glycoprotein G of vesicular stomatitis virus implanted into the plasma membrane and then internalized for different times at 37 degrees C. The corresponding endosomal fractions were immunoisolated using the cytoplasmic domain of the G protein as antigen. These fractions were then used in an in vitro assay to quantify the efficiency of fusion between endosomal vesicles. To identify the vesicular partners of the fusion, these in vitro studies were combined with in vivo biochemical and morphological experiments. Internalized molecules were delivered to early endosomal elements, which corresponded to a network of tubular and tubulovesicular structures. Rapid recycling back to the plasma membrane and routing to late stages of the pathway occurred from these early endosomal elements. These elements exhibited a high and specific fusion activity with each other in vitro, suggesting that individual elements of the early endosomal compartment interact with each other in vivo. After their appearance in the early endosome, the molecules destined to be degraded were observed at the next stage of the pathway in distinct spherical vesicles (0.5 micron diam) and then in late endosomes and lysosomes. When the microtubules were depolymerized with nocodazole, endocytosis proceeded as in control cells. However, internalized molecules remained in the spherical vesicles and did not appear in late endosomes or lysosomes. These spherical vesicles had relatively little fusion activity with each other or with early endosomal elements in vitro. Our observations suggest that the spherical vesicles mediate transport between the early endosome and late endosomes and that this process requires intact microtubules.     

Characterization of Amoeba proteus myosin VI immunoanalog

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.     

Determining the cellular function of myosin VI

A report on work presented at the 39th Annual Meeting of the American Society for Cell Biology, Washington DC, December 11-15, 1999     

Motor protein independent binding of endocytic carrier vesicles to microtubules in vitro

We have established an in vitro assay to characterize the binding of endocytic carrier vesicles to microtubules. Magnetic beads coated with microtubules were used as an affinity matrix. A fraction from nocodazole-treated cells enriched in endocytic carrier vesicles, labeled with internalized horseradish peroxidase, was used in the binding experiments. Binding of the endocytic carrier vesicles to microtubules in vitro was cytosol-dependent. This activity of cytosolic factors was saturable, heat-sensitive, and insensitive to N-ethyl-maleimide. Binding was sensitive to GTP and ATP. Addition of neuronal microtubule-associated proteins completely abolished binding of the endocytic organelles to microtubules. This binding was independent of the cytosolic microtubule-based motor proteins kinesin and cytoplasmic dynein, since cytosol depleted of these proteins remained fully active. Microtubule-binding proteins from HeLa cells, however, stimulated the interaction of endocytic carrier vesicles with microtubules. Trypsinized vesicles could no longer bind to microtubules in the presence of cytosol. These results suggest that cytosolic microtubule-binding proteins, other than the known microtubule-based motor proteins, as well as membrane proteins are involved in the nucleotide-dependent interaction of endocytic carrier vesicles with microtubules.     

Characterization of myosin V binding to brain vesicles

Myosin II and V are important for the generation and segregation of subcellular compartments. We observed that vesicular myosin II and V were associated with the protein scaffolding of a common subset of vesicles by density sedimentation, electron microscopy, and immunofluorescence. Solubilization of either myosin II or V was caused by polyphosphates with the following efficacy at 10 mM: for myosin II ATP-Mg(2+) = ATP = AMP-PNP (5'-adenylyl imidodiphosphate) > pyrophosphate = tripolyphosphate > tetrapolyphosphate = ADP > cAMP = Mg(2+); and for myosin V pyrophosphate = tripolyphosphate > ATP-Mg(2+) = ATP = AMP-PNP > ADP = tetrapolyphosphate > cAMP = Mg(2+). Consequently, we suggest solubilization was not an effect of phosphorylation, hydrolysis, or disassociation of myosin from actin filaments. Scatchard analysis of myosin V binding to stripped dense vesicles showed saturable binding with a K(m) of 10 nM. Analysis of native vesicles indicates that these sites are fully occupied. Together, these data show there are over 100 myosin Vs/vesicle (100-nm radius). We propose that polyphosphate anions bind to myosin II and V and induce a conformational change that disrupts binding to a receptor.     

Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.     

Membrane lipids and proteins as modulators of urothelial endocytic vesicles pathways

The increased studies on urinary bladder umbrella cells as an important factor for maintaining the permeability barrier have suggested new pathways for the discoidal/fusiform endocytic vesicles which is one of the main features of the umbrella cells. The biological role of these vesicles was defined, for many years, as a membrane reservoir for the umbrella cell apical plasma membrane which are subject to an increased tension during the filling phase of the micturition cycle and, therefore, the vesicles are fused with the apical membrane. Upon voiding, the added membrane is reinserted via a non-clathrin or caveolin-dependant endocytosis thereby restoring the vesicle cytoplasmic pool. However, in the last decade, new evidence appeared indicating alternative pathways of the endocytic vesicles different than the cycling process of exocytosis/endocytosis. The purpose of this review is to analyze the molecular modulators, such as membrane lipids and proteins, in the permeability of endocytic vesicles, the sorting of endocytosed material to lysosomal degradation pathway and recycling of both membrane and fluid phases.