Fusion Of Endocytic Vesicles Research Articles

3] Detection of endocytic vesicle fusion in Vitro, using assay based on avidin-biotin association reaction

Publisher Summary Endocytosis and phagocytosis are the two primary mechanisms employed by eukaryotic cells to internalize macromolecules from the extracellular milieu. The most comprehensively studied endocytic pathway is receptor-mediated endocytosis via clathrin-coated pits and subsequent transport to lysosomes. In the early endosome, ligandreceptor dissociation and sorting occur. Soon afterward, receptors recycle to the plasma membrane, while free ligand progresses to another population of vesicles termed, “late endosomes.” Contrary to endocytosis, phagocytosis is a function of specialized cells. In vertebrates, mononuclear macrophages and polymorphonuclear granulocytes are the primary phagocytes. The biochemical mechanisms that regulate phagolysosome biogenesis are very complex. As evidenced by a number of investigations, both membrane fusion and membrane recycling play important roles in this process. Therefore, to better understand the mechanisms employed by phagosomes to process and traffic internalized material, development of probes and cell-free assays.

Characterization of endocytosis in intact cells by quantitative fluorescence microscopy

Endocytosis, the process by which extracellular macromolecules are taken into the cell is particularly amenable to quantitative analysis using fluorescence techniques. Fluorescently-labeled proteins and other macromolecules are taken up by cells via receptor-mediated endocytosis or fluid phase pinocytosis. Recently, fluorescent lipid probes have also been synthesized which label the endocytic pathway, allowing observation of lipid membrane traffic.Digital imaging allows the manipulation of images, background and autofluorescence subtractions, and fluorescence intensity measurements of individual endocytic compartments. Image intensification microscopy and digital image processing have been used extensively for studies of endocytosis, including the measurements of endosome pH, and the identification and quantitation of endocytic vesicle fusion. Recently, pathways taken by endocytosed fluorescent molecules have been followed using the confocal microscope, adding a powerful tool to investigate questions about endocytosis which were very difficult to study with conventional, full-field microscopy techniques.

Changes in macromolecular synthesis of gypsy moth cell line IPLB-Ld652Y induced by Autographa californica nuclear polyhedrosis virus infection

The aberrant replication of the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) in the Lymantria dispar cell line IPLB-Ld652Y was used as a model system for the investigation of factors regulating baculovirus host specificity. A previous study of this system indicates that viral gene expression in infected cells is extremely attenuated and subsequently all cellular and viral protein synthesis is inhibited. In the present study, infection of IPLB-Ld652Y cells with AcMNPV photochemically inactivated in situ resulted in a rapid reduction in cell mitotic indices and cell growth, as well as inducing a series of distinct morphological changes in these cells. At the molecular level, infection with inactivated virus, followed by pulse labelling with [3H]thymidine, resulted in a rapid [0 to 2 h post-infection (p.i.)] and permanent inhibition of host cellular DNA synthesis. Assays of cellular DNA polymerases in isolated IPLB-Ld652Y nuclei confirmed the reduction in cellular DNA synthesis observed in intact cells and indicated an initial (0 to 2 h p.i.) reduction in the activity of aphidicolin-sensitive DNA polymerases. Activity of all cellular DNA polymerases was inhibited at later times p.i. Host cell protein synthesis was completely inhibited after 48 h p.i. Treatment of inactivated virus and virus-infected cells with various chemical and physical factors (i.e. pH and temperature) or lysosomotropic agents revealed that virus entry into cells and fusion of endocytic vesicles (containing virus) with lysosomes were essential for suppression of cellular macromolecular synthesis. The possible involvement of structural components of the AcMNPV virion in these effects is discussed.

Characterization of the mechanism of endocytic vesicle fusion in vitro.

A cell-free assay to monitor receptor-mediated endocytic processes has been developed that uses biotinylated transferrin and avidin-linked beta-galactosidase as receptor-associated and fluid-phase probes, respectively (Wessling-Resnick, M., and Braell, W. A. (1990) J. Biol. Chem. 265, 690-699). The fusion of vesicles from heterologous sources can be detected in this assay: endocytic vesicles from K562 cells (a human cell line) will fuse with vesicles from Chinese hamster ovary cells. Fusion between endocytic vesicles is inhibited upon treatment with N-ethylmaleimide but can be restored by the addition of untreated cytosol from either cell type. The in vitro fusion reaction is also inhibited by the nonhydrolyzable nucleotide analogs guanosine 5'-(3-thiotriphosphate) (GTP gamma S) and adenosine 5'-(3-thiotriphosphate) (ATP gamma S). Other nonhydrolyzable guanine nucleotides are found to inhibit the in vitro reaction in the following order of potency: GTP gamma S greater than 5'-guanylyl imidodiphosphate (GTP-PNP) greater than alpha,beta-methylene GTP (GTP-PCP). The inhibitory effects of the nonhydrolyzable analogs of GTP and ATP are not additive. Moreover, excess GTP relieves the inhibition by GTP gamma S more than it relieves the inhibition by ATP gamma S, while excess ATP preferentially alleviates ATP gamma S (not GTP gamma S) inhibition. These properties suggest that the two nucleotides exert their effects at distinct points in the fusion process. Although micromolar levels of excess Ca2+ also inhibit vesicle fusion, the inhibition exerted by GTP gamma S appears to proceed via a pathway independent of the divalent cation. The GTP gamma S-sensitive step in endocytic vesicle fusion is found to occur at a mechanistic stage prior to and distinct from the N-ethylmaleimide-sensitive step of the reaction. This situation permits the accumulation of a membrane vesicle intermediate in the presence of GTP gamma S; subsequent incubation of these vesicles with cytosol and GTP restores their fusion competence. Characteristics of in vitro endocytic vesicle fusion suggest that similarities exist with steps of the fusion mechanism involved with membrane traffic events of the secretory pathway.

The sorting and segregation mechanism of the endocytic pathway is functional in a cell-free system.

A cell-free system which reconstitutes early stages of receptor-mediated endocytosis has been developed, based on detection of the association between avidin-beta-galactosidase (Av beta Gal) and biotin-transferrin (B-Tf). Initially, Av beta Gal (a fluid-phase marker) and B-Tf (receptor-bound) are internalized and delivered to a common endosomal compartment in vivo and in vitro. Subsequently, these two probes enter divergent intracellular pathways: Av beta Gal is sorted from the endosome and directed for delivery to lysosomes, whereas B-Tf is segregated away from the fluid-phase marker, remaining bound to the transferrin receptor for return to the cell surface. Using the avidin-biotin association reaction to monitor the co-localization of these two probes, we have been able to reconstruct this sorting and segregation process in a cell-free system. The in vitro reaction is time-, temperature-, and ATP-dependent, and is not affected by NH4Cl; cell-free segregation of the two probes is also sensitive to N-ethylmaleimide. As these characteristics are also properties of in vitro endocytic vesicle fusion, it is likely that the latter event is a prerequisite for the sorting and segregation process. Both the in vivo and in vitro sorting of Av beta Gal and B-Tf to their respective and distinct destinations can be followed by subcellular fractionation on Percoll gradients. Our observations provide the first evidence that the cellular mechanism to identify, sort, and sequester endocytosed material can be reconstituted in a cell-free system.

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.

Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport.

In reconstitution studies N-ethylmaleimide, a sulphydryl alkylating reagent, inhibits both fusion of endocytic vesicles and vesicular transport in the Golgi apparatus. We show here that the same N-ethylmaleimide-sensitive factor that catalyses the vesicle-mediated transport within Golgi stacks is also required for endocytic vesicle fusion. Thus, it is likely that a common mechanism for vesicle fusion exists for both the secretory and endocytic pathways of eukaryotic cells.

Pathway and kinetics of vitellogenin‐gold internalization in the Xenopus oocyte

After in vitro incubation of Xenopus oocytes with vitellogenin (VTG)-gold conjugate, the gold particles are distributed on the whole plasma membrane. Their concentration in coated pits still occurs at 0 degrees C. At +20 degrees C the label quickly (30 sec) appears in multi-vesicular endosomes (MVE) which segregate together with primary endocytic vesicles into distinct clusters below the plasma membrane. From this step up to crystallization of the yolk platelets, the gold particles stay in the same compartment. During 5.5 h the label progressively increases along the MVE membrane, first (1.5 h) by fusion of primary endocytic vesicles with consecutively enlarging endosomes, then (4 h) by decreasing of the MVE membrane. As concerns the yolk platelet formation, concentration of primordial yolk platelets (PYP) occurs at 5.5 h from the incubation onset, the labeling of preexisting yolk platelets starts at 7 h, while crystallization of PYP begins only after 12-13 h. Our results indicate that VTG receptors are not preclustered in coated pits and their lateral translation is not inhibited at 0 degrees C. The yolk protein processing takes place within one compartment only. The VTG condensation begins with a long concentration phase of receptor-VTG complexes still integrated in the endosome membrane. It occurs in MVE by: i) a repeated fusion of primary endocytic vesicles; ii) removing part of the endosome membrane by internal vesiculation. Fusion between endosomes occurs only after VTG has dissociated from its receptors and VTG dissociates only when when the density of the VTG-receptor complexes in the endosome membrane is sufficient. Crystallization begins after a 7-8 h delay. The endosome migration into the oocyte is also controlled by the binding of VTG to its receptors. Our results also demonstrate that binding of VTG colloidal gold modifies neither the vitellogenic pathway nor the duration of the vitellogenin internalization. However when vitellogenin is bound to colloidal gold, dissociation of ligand-receptor complexes is delayed because the amount of ligand in the incubation medium is necessarily low.

Intracellular formation of calcium concretions by phagocytic cells in freshwater mussels

The gills of freshwater unionid mussels contain large accumulations of extracellular calcium phosphate concretions. Connective tissue cells located in specific areas in the gills of these animals are the site of concretion formation. The cell type responsible for concretion formation appears to differentiate from an undifferentiated precursor cell by accumulating endocytic vesicles and producing amorphous membrane-bound granules from active rough endoplasmic reticulum and Golgi apparatus. In the most mature of the cells, these granules occupy most of the cytoplasm. Concretions are initiated within the amorphous granules as observed using stereo-pair transmission electron microscopy. These connective tissue cells exhibit phagocytosis, and can ingest India ink particles and colloidal gold. Colloidal gold is internalized through an endocytic mechanism within vesicles. Gold can be observed in the amorphous granules, implying fusion of such endocytic vesicles with the amorphous granules. While the exact mechanism of concretion formation is not understood, the cellular site of formation has been identified. Further, protein synthetic activity as well as endocytic activity appear necessary for concretion production.

Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system.

We have used defined subcellular fractions to reconstitute in a cell-free system vesicle fusions occurring in the endocytic pathway. The endosomal fractions were prepared by immuno-isolation using as antigen an epitope located on a foreign protein, the transmembrane glycoprotein G (G-protein) of vesicular stomatitis virus. The G-protein was first implanted in the cell plasma membrane and subsequently endocytosed for 15 to 30 min at 37 degrees C. The endosomal fractions were immuno-isolated on a solid support using as antigen the cytoplasmic domain of the G-protein in combination with a specific monoclonal antibody. For comparative studies the plasma membrane was immuno-isolated from cells in the absence of G internalization with a monoclonal antibody against the exoplasmic domain of the G-protein. The immuno-isolated endosomal vesicles contained 70% of horseradish peroxidase internalized in the endosome fluid phase, exhibited an acidic luminal pH as shown by acridine orange fluorescence and differed in their protein composition from the immuno-isolated plasma membrane fraction. The fusion of endocytic vesicles originating from different stages of the pathway was studied in a cell-free assay using both a bio-chemical and a morphological detection system. These well defined endosomal vesicles were immuno-isolated with the G-protein on the solid support and provided the recipient compartment of the fusion (acceptor). They were mixed with a post-nuclear supernatant containing endosomes loaded with exogenous lactoperoxidase (donor) at 37 degrees C. Fusion delivered the donor peroxidase to the lumen of acceptor vesicles permitting fusion-specific iodination of the G-protein itself. The fusion of vesicles required ATP and was detected only with an endosomal fraction prepared after internalization of the G-protein for 15 min at 37 degrees C but not with a plasma membrane or with an endosomal fraction prepared after 30 min G-protein internalization.

Influence of proteolytic enzymes and calcium-binding agents on nuclear and cell surface topography.

Transmission electron microscopy was used to study the effects of proteolytic enzymes (collagenase, trypsin, clostripain), the calcium chelator ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), and the calcium ionophore A 23187 on substrate adhesion and fine structure of chondrocytes and fibroblasts. Monolayer-cultured cells responded to treatment with the proteolytic enzymes followed by EGTA or A 23187 by rounding and detaching from the substrate. This was accompanied by the formation of a microvillous surface, deep nuclear folds, and numerous cytoplasmic vacuoles. Labeling experiments with colloidal thorium dioxide indicated that the vacuoles were formed by endocytosis and fusion of endocytic vesicles with preexisting lysosomes. To a variable extent, similar changes were produced by trypsin or EGTA alone. The cells regained their normal fine structure after withdrawal of the reagents and when seeded onto a substrate. In suspension culture, recovery was incomplete; the cells retained a rounded shape and an increased number of cytoplasmic vacuoles. The results suggest that changes in plasma membrane composition and its permeability to calcium represent the primary signal for cell rounding and detachment. The cellular mechanisms responsible for the associated folding of the nuclear envelope and the cell surface remain unidentified. Nevertheless, this is believed to represent a means of handling of excess membrane during sudden transition from a flattened to a rounded shape. Membrane stored in folds and vacuoles is reutilized when the cells reattach and spread out on a substrate.

Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line.

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.

Flow cytofluorometric analysis of insulin binding and internalization by swiss 3T3 cells

The binding of a fluorescein-isothiocyanate derivative of insulin to Swiss 3T3 cells was measured by flow cytometry. The kinetics of the subsequent internalization were also measured; at a concentration of 1 microM labeled insulin approximately 25% of the internalization was insulin-specific. The kinetics of endocytosis were contrasted to those of fluorescent derivatives of histone and dextran. In addition, the fusion of endocytic vesicles containing insulin or dextran with lysosomes was detected by measuring the pH-dependent increase in fluorescein fluorescein fluorescence caused by the addition of chloroquine. The application of these results to the analysis of growth control by insulin and related hormones is discussed.

Autoradiographic localization of the sites of uptake, cellular transport, and catabolism of low density lipoproteins in the liver of normal and estrogen-treated rats.

The hepatic uptake and catabolism of low density lipoproteins are stimulated severalfold in rats treated with large amounts of 17alpha-ethinylestradiol. To determine the sites within the liver at which these processes occur, (125)I-labeled human low density lipoproteins were injected intravenously into intact control and estradiol-treated rats or added to perfusates of their isolated livers. The livers were fixed by perfusion and processed for light and electron microscopic autoradiography. Distribution of autoradiographic silver grains was estimated qualitatively in light micrographs and quantitatively in electron micrographs. Many more silver grains were seen in livers from estradiol-treated than from control rats, but the processing of labeled low density lipoprotein was indistinguishable. Three minutes after intravenous injection or perfusion of livers, the grains were concentrated over the microvillous surface of parenchymal cells bordering the space of Disse. Many of these grains were within two half-distances from endocytic pits. Only 5-15% of the grains were seen over endothelial and Kupffer cells. Silver grains were also observed over vesicles beneath the plasma membrane whose size and shape suggested that they were derived from fusion of endocytic vesicles. By 15 min, grains were predominantly located in structures like multivesicular bodies in the region of the GERL (Golgi complex-endoplasmic reticulum-lysosomes) near the bile canaliculi. These bodies were packed with small vesicle-like structures and a few larger vesicles, the latter possessing a unit membrane. Between 15 and 30 min, when proteolysis of low density lipoproteins is known to begin, the initially clear matrix of the multivesicular body-like structures became dark and the structures frequently had a dense tail-like appendage. At the same time, silver grains began to appear over secondary lysosomes. These and other results indicate that the hepatic uptake of low density lipoproteins that is stimulated in rats given large amounts of estradiol follows a pathway that closely resembles that of the well-defined "LDL receptor" in cultured cells. In the liver these lipoproteins appear to be transported in endocytic vesicles; the vesicles fuse to form multivesicular body-like structures that acquire lysosomal enzymes and are converted to secondary lysosomes as the lipoproteins are degraded.

Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes.

Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.

Microperoxidase uptake into the rat choroid plexus epithelium

Uptake, transport, and diffusion of the heme-peptide tracer microperoxidase (MP; MW 1900) in the rat choroid plexus were studied. MP was perfused ventriculo—cisternally or intravenously. The plexuses were fixed by ventriculo—cisternal perfusion of aldehydes, and tissue sections were incubated in a diaminobenzidine—H 2 O 2 medium containing imidazole at pH 8.8. After intravenous administration MP was found in the connective tissue of the plexus. Basement membranes were heavily labeled, while intercellular spaces between epithelial cells exhibited only a little MP. Some micropinocytic uptake of MP into vacuoles and lysosomes of the epithelial cells occurred. After ventriculo-cisternal perfusion of MP, it was micropinocytozed and incorporated into numerous vacuoles, multivesicular bodies, and dense bodies. Labeling of elongated cisterns (ER cisterns, or cisterns formed by fusion of endocytic vesicles) also occurred. Apical tight junctions between choroid plexus epithelial cells were never penetrated by MP. The intercellular spaces between epithelial cells, the connective tissue between epithelium and endothelium, and the blood vessels were, in general, devoid of MP, although diffusion of MP between ependymal cells into the extracellular space of the neuropil close to the base of the plexus was pronounced, as evaluated from the density of reaction product. No MP could be located in the kidney tubules of rats exposed ventriculo—cisternally to MP. The observations indicate that the choroid plexus is involved in absorption of molecules—also smaller than proteins—from the cerebrospinal fluid (csf). No significant diffusion of MP via ependyma and neuropil (the “functional leak”) into the plexus occurred, and no support of a transepithelial vesicular csf-to-blood transport of MP was obtained. This discrepancy in relation to previous horseradish peroxidase experiments is discussed.