Endosome-to-cytosol transport of viral nucleocapsids.

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns3P) signalling via the PtdIns3P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns3P and their effectors.

Regulation of intracellular membrane transport.

A number of proteins that are necessary for membrane transport have been identified using cell-free assays and yeast genetics. Although our knowledge of transport mechanisms remains limited, common themes are clearly emerging. In particular, specific GTP-binding proteins appear to be involved, not only at all steps of membrane traffic but also at more than one check-point within each step. The ordered sequence of events occurring during vesicle formation, targeting and fusion may be regulated in a stepwise manner by specific GTP-dependent switches, which act as modular elements of the transport mechanism.

Membrane transport in the endocytic pathway.

Despite controversies and debates, some fundamental properties of endosomes become apparent when comparing results from in vivo and in vitro strategies used to study endosomal membrane traffic. In addition, recent studies are starting to unravel the complex organization of early endosomes, in particular along the route followed by recycling receptors.

Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport.

The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC.

Jamming the endosomal system: lipid rafts and lysosomal storage diseases.

Some lysosomal storage diseases result from the accumulation of lipids in degradative compartments of the endocytic pathway. Particularly striking is the example of the Niemann-Pick (NP) syndrome. NP syndromes types A and B are characterized by the accumulation of sphingomyelin, whereas cholesterol typically accumulates in NP type C. These two different lipids, sphingomyelin and cholesterol, are normal constituents of specific lipid microdomains called rafts. Because accumulation of raft lipids is observed not only in NP diseases but also in many other lipidoses, we forward the hypothesis that lysosomal storage diseases can be caused by the accumulation of lipid rafts in late endosomes/lysosomes.

Annexins in membrane traffic.

Annexins have long been though to be involved in exocytosis, possibly by helping to create close interactions between membranes destined to undergo fusion. In this article, we examine recent observations that implicate annexins in three different steps of the endocytic pathway, suggesting that annexins may be universal modulators of membrane trafficking.

The arguments for pre-existing early and late endosomes.

The past decade has seen the elucidation of many of the events and processes responsible for receptor-mediated endocytosis. However, a fundamental question about the endocytic pathway remains unresolved: do early endosomes mature into late endosomes, or are these two distinct and pre-existing cellular organelles? General opinion tends to favour the former possibility, to the point where one poster session at the recent American Society for Cell Biology meeting was entitled 'Maturation of Endosomes'. This article draws together new data arguing in favour of pre-existing early and late endosomes, between which transport occurs by vesicle budding and fusion.

Cytoplasmic dynein-dependent vesicular transport from early to late endosomes.

We have used an in vitro fusion assay to study the mechanisms of transport from early to late endosomes. Our data show that the late endosomes share with the early endosomes a high capacity to undergo homotypic fusion in vitro. However, direct fusion of early with late endosomes does not occur. We have purified vesicles which are intermediates during transport from early to late endosomes in vivo, and analyzed their protein composition in two-dimensional gels. In contrast to either early or late endosomes, these vesicles do not appear to contain unique proteins. Moreover, these vesicles undergo fusion with late endosomes in vitro, but not with each other or back with early endosomes. In vitro, fusion of these endosomal vesicles with late endosomes is stimulated by polymerized microtubules, consistent with the known role of microtubules during early to late endosome transport in vivo. In contrast, homotypic fusion of early or late endosomes is microtubule-independent. Finally, this stimulation by microtubules depends on microtubule-associated proteins and requires the presence of the minus-end directed motor cytoplasmic dynein, but not the plus-end directed motor kinesin, in agreement with the microtubule organization in vivo. Our data strongly suggest that early and late endosomes are separate, highly dynamic organelles, which are connected by a microtubule-dependent vesicular transport step.

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.

PI3P signaling regulates receptor sorting but not transport in the endosomal pathway.

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

Scanning electron microscope-analysis of the protrusions (knobs) present on the surface of Plasmodium falciparum-infected erythrocytes.

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs X micron-2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs X micron-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.

Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes.

In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in epsilon coatomer protein (epsilonCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although epsilonCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of epsilonCOP, we find that a subcomplex of beta, beta', and zetaCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPgammaS and sensitivity to the endosomal pH. Previous studies showed that gamma and deltaCOP are not found on endosomes. However, alphaCOP, which is normally present on endosomes, is no longer recruited when epsilonCOP is missing. In contrast, all COP subunits, except obviously epsilonCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas beta, beta', and zetaCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of alpha and/or epsilonCOP.

An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes.

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.

Endocytosis in filter-grown Madin-Darby canine kidney cells.

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three-dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest.

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

Annexin II is a major component of fusogenic endosomal vesicles.

We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two-dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.

Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

Subcellular fractionation of tissue culture cells.

Subcellular fractionation has two major steps, (1) the homogenization of the cells and (2) the subsequent separation of the organelles. The homogenization step is discussed with reference to the problems encountered using tissue culture cells. Promising techniques for the isolation of specific compartments are illustrated using the isolation of the endosomal compartment as the example.

Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components.

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.