Fusion accessibility of endocytic compartments along the recycling and lysosomal endocytic pathways in intact cells

A fluorescence assay developed for the quantitation of intracellular fusion of sequentially formed endocytic compartments (Salzman, N. H., and F. R. Maxfield. 1988 J. Cell Biol. 106:1083-1091) has been used to measure the time course of endosome fusion accessibility along the recycling and degradative endocytic pathways. Transferrin (Tf) was used to label the recycling pathway, and alpha2-macroglobulin (alpha 2 M) was used to label the lysosomal degradative pathway. Along the degradative pathway, accessibility of vesicles containing alpha 2M to fusion with subsequently formed endocytic vesicles decreased with apparent first order kinetics. The t12 for the loss of fusion accessibility was approximately 8 min. The behavior of Tf is more complex. Initially the fusion accessibility of Tf decayed rapidly (t1/2 less than 3 min), but a constant level of fusion accessibility was then observed for 10 min. This suggests that Tf moves through one fusion accessible endosome rapidly and then enters a second fusion accessible compartment on the recycling pathway. At 18 degrees C, fusion of antifluorescein antibodies (AFA) containing vesicles with F-alpha 2M was observed when the interval between additions was 10 min. However, if the interval was increased to 1 h, no fusion with incoming vesicles was observed. These results identify the site of F-alpha 2M accumulation at 18 degrees C as a prelysosomal late endosome that no longer fuses with newly formed endosomes since no delivery to lysosomes is observed at this temperature.     

Plasma membrane-derived vesicles containing receptor-ligand complexes are fusogenic with early endosomes in a cell-free system

Receptor-mediated endocytosis involves the transport of receptor-ligand complexes from the cell surface to an intracellular endocytic compartment. This study shows that plasma membrane-derived vesicles containing receptor-bound ligands (e.g. aggregated anti-dinitrophenol (DNP) IgG bound to Fc receptors) fuse with early endosomes containing DNP-beta-glucuronidase in a cell-free system. Plasma membrane vesicles were generated by homogenization of cells that had been allowed to bind ligands at 4 degrees C. Fusion between vesicles containing the two probes was assessed by (i) the formation of anti-DNP IgG-DNP-beta-glucuronidase complexes and (ii) the colocalization within closed vesicles of two different sizes of colloidal gold coated with ligands. Fusion required ATP, cytosol, and KCl. The requirements were similar to those described for endosome-endosome fusion in in vitro systems. Mild trypsinization of vesicles prior to their addition to the assay inhibited fusion. When DNP-beta-glucuronidase was chased into more mature endocytic compartments, fusion was not observed. The results indicate that cell surface regions involved in receptor-mediated endocytosis are capable of fusing to early endosomes. This fusion event may constitute the first step in the transport of ligands to an intracellular endocytic compartment.     

In vitro clustering and multiple fusion among macrophage endosomes

Early steps of receptor-mediated endocytosis appear to require the fusion of endosomes with each other. Recently, these fusion events have been reconstituted in vitro using vesicle preparations from J774 macrophages which have internalized ligands via the mannose receptor (Diaz, R., Mayorga, L., and Stahl, P. (1988) J. Biol. Chem. 263, 6093-6100). The present studies indicate that endosomes first form clusters when incubated under fusogenic conditions. Aggregation state was determined by electron microscopy using vesicles containing ligand-coated colloidal gold of different sizes previously internalized via the mannose receptor. Aggregation required cytosol and ATP. Afterwards, the limiting membranes of the vesicles composing these aggregates undergo multiple fusion and bring about the formation of large diameter vesicles that maintained the same density as endosomes when analyzed by Percoll gradient sedimentation. These large diameter vesicles were no longer fusogenic in the fusion assay. Multiple fusion was determined morphologically by the co-localization of three different size colloidal gold vesicles inside endocytic vesicles and biochemically by the fusion-dependent formation of triple immune complexes between three endocytic ligands internalized by receptor-mediated endocytosis: anti-dinitrophenol mouse IgG and dinitrophenol-derivatized beta-glucuronidase, ligands for the mannose receptor, and aggregated rabbit anti-mouse IgG, a ligand for the macrophage Fc receptor.     

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.     

Phospholipid diversity: correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called "fusion proteins". This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

GD1b-Derived Gangliosides Modulate Fc��RI Endocytosis in Mast Cells

The role of the mast cell–specific gangliosides in the modulation of the endocytic pathway of FcεRI was investigated in RBL-2H3 cells and in the ganglioside-deficient cell lines, E5 and D1. MAb BC4, which binds to the α subunit of FcεRI, was used in the analysis of receptor internalization. After incubation with BC4-FITC for 30 min, endocytic vesicles in RBL-2H3 and E5 cells were dispersed in the cytoplasm. After 1 hr, the endocytic vesicles of the RBL-2H3 cells had fused and formed clusters, whereas in the E5 cells, the fusion was slower. In contrast, in D1 cells, the endocytic vesicles were smaller and remained close to the plasma membrane even after 3 hr of incubation. When incubated with BC4-FITC and subsequently imunolabeled for markers of various endocytic compartments, a defect in the endocytic pathway in the E5 and D1 cells became evident. In the D1 cells, this defect was observed at the initial steps of endocytosis. Therefore, the ganglioside derivatives from GD1b are important in the endocytosis of FcεRI in mast cells. Because gangliosides may play a role in mast cell–related disease processes, they provide an attractive target for drug therapy and diagnosis.     

Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages

Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome-lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.     

Phospholipid diversity: Correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called “fusion proteins”. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Phosphatidylethanol counteracts calcium-induced membrane fusion but promotes proton-induced fusion

The susceptibility of phosphatidylethanol-containing lipid vesicles towards Ca2+- and proton-induced fusion has been investigated, using a system of interacting vesicles. The results show that phosphatidylethanol-rich vesicles are quite resistant to Ca2+-induced fusion while being highly sensitive to proton-induced fusion. Inclusion of phosphatidylethanol was also found to promote and inhibit, respectively, the proton-induced and Ca2+-induced fusion of bilayer vesicles containing also phosphatidylethanolamine and either phosphatidylserine or phosphatidic acid. Thus, phosphatidylethanol affected Ca2+- and proton-induced fusion in opposite directions, in contrast to the naturally occurring anionic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol, which affect the sensitivity to Ca2+- and H+-induced fusion in the same direction. However, the fusion competence of phosphatidylethanol vesicles in response to both Ca2+ and H+ was inversely related to the apparent thickness of the polar headgroup layer, determined by using lectin-glycolipid interaction as a steric probe, as previously found for vesicles containing naturally occurring anionic phospholipids.     

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.     

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.     

beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases

Rab GTPases are recognized as critical regulatory factors involved in vesicular membrane transport and endosomal fusion. For example, Rab5 directs the transport and fusion of endocytic vesicles to and with early endosomes, whereas Rab4 is thought to control protein trafficking from early endosomes back to the plasma membrane. In the present study, we investigated the role of Rab5 and Rab4 GTPases in regulating the endocytosis, intracellular sorting, and the plasma membrane recycling of the beta(2)AR. In cells expressing the dominant-negative Rab5-S34N mutant, beta(2)AR internalization was impaired, and beta(2)AR-bearing endocytic vesicles remained in either close juxtaposition or physically attached to the plasma membrane. In contrast, a constitutively active Rab5-Q79L mutant redirected internalized beta(2)AR to enlarged endosomes but did not prevent beta(2)AR dephosphorylation and recycling. The expression of either wild-type Rab4 or a Rab4-N121I mutant did not prevent beta(2)AR dephosphorylation. However, the dominant-negative Rab4-N121I mutant blocked beta(2)AR resensitization by blocking receptor recycling from endosomes back to the cell surface. Our data indicate that, in addition to regulating the intracellular trafficking and fusion of beta(2)AR-bearing endocytic vesicles, Rab5 also contributes to the formation and/or budding of clathrin-coated vesicles. Furthermore, beta(2)AR dephosphorylation occurs as the receptor transits between Rab5- and Rab4-positive compartments.     

Myo6 facilitates the translocation of endocytic vesicles from cell peripheries

Immunolocalization studies in epithelial cells revealed myo6 was associated with peripherally located vesicles that contained the transferrin receptor. Pulse-chase experiments after transferrin uptake showed that these vesicles were newly uncoated endocytic vesicles and that myo6 was recruited to these vesicles immediately after uncoating. GIPC, a putative myo6 tail binding protein, was also present. Myo6 was not present on early endosomes, suggesting that myo6 has a transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome.     

In vitro fusion of endosomes following receptor-mediated endocytosis

Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.     

Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum.

An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP-derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O-(thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway.     

The vacuolar import and degradation pathway merges with the endocytic pathway to deliver fructose-1,6-bisphosphatase to the vacuole for degradation

The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the epsilon-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Deltavph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.     

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.     

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.     

Incorporation of semi-fluorinated alkanes in the bilayer of small unilamellar vesicles of phosphatidylserine: impact on fusion kinetics

Semi-fluorinated alkanes C(n)F(2n+1)C(m)H(2m+1) (FnHm) can be co-dispersed with standard phospholipids to form 'fluorinated' vesicles, i.e. vesicles with an internal fluorinated film within their bilayer membrane. This paper reports the effect of the presence of such FnHm diblocks in phosphatidylserine (PS)-based small unilamellar vesicles (SUVs) on their kinetics of fusion. Fusion was induced by calcium ions and monitored by the terbium/dipicolinic acid assay. The diblocks were composed of a 10-carbon long linear hydrocarbon segment and of a linear fluorocarbon segment of four, six or eight carbon atoms. We found that the incorporation of FnHm in the PS membrane considerably modifies the kinetics of the process of fusion, with Ca(2+) concentration having a much more limited effect on the fluorinated vesicles. Both the rates of fusion and the rates of release of the internal content, as evaluated by the release of 5,6-carboxyfluorescein, were much lower for the fluorinated SUVs than for those based on phosphatidylserine alone, the highest effect being obtained for F6H10 with a 10 times slower rate of fusion and a 40-fold reduction in the release of content. FnHm molecules are proposed to have a dual action: by hindering fusion and release by creating an inert, hydrophobic and lipophobic fluorinated film in the core of the membrane, and by stabilizing the membrane by increasing van der Waals interactions in the hydrocarbon region.     

Comparison of two liposome fusion assays monitoring the intermixing of aqueous contents and of membrane components

Divalent cation-induced fusion of large unilamellar vesicles (approx. 0.1 micron diameter) made of phosphatidylserine (PS) or phosphatidylglycerol (PG) has been studied. Intermixing of aqueous contents during fusion was followed by the Tb/dipicolinic acid fluorescence assay, and intermixing of membrane components by resonance energy transfer between fluorescent lipid probes. Both assays gave identical threshold concentrations for Ca2+, which were 2 mM for PS and 15 mM for PG. The dependencies of the initial rate of fusion on the concentration of PG vesicles determined by either assay were identical, the order of this dependence being 1.2 in the concentration range of 5-200 microM lipid. For PS liposomes, this order was found to be 1.5 in the fluorescent lipid assay. No leakage of contents was detected during the fusion of PG vesicles. Mg2+ inhibited the Ca2+-induced fusion of PS vesicles, but did not cause any fusion by itself, consistent with previous results with the Tb/dipicolinic acid assay.