The role of ADP-ribosylation factor and phospholipase D in adaptor recruitment.

AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTPgammaS stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093-1105). We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTPgammaS-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTPgammaS loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTPgammaS. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTPgammaS or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.

The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.

We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome.

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH(2)-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of gamma-adaptin. However, unlike gamma-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the gamma-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.

Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.

Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37 degrees C, late endosome-lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

A novel adaptor-related protein complex.

Coat proteins are required for the budding of the transport vesicles that mediate membrane traffic pathways, but for many pathways such proteins pathways, but for many pathways such proteins have not yet been identified. We have raised antibodies against p47, a homologue of the medium chains of the adaptor complexes of clathrin-coated vesicles (Pevsner, J., W. Volknandt, B.R. Wong, and R.H. Scheller. 1994. Gene (Amst.). 146:279-283), to determine whether this protein might be a component of a new type of coat. p47 coimmunoprecipitates with three other proteins: two unknown proteins of 160 and 25 kD, and beta-NAP, a homologue of the beta/beta'-adaptins, indicating that it is a subunit of an adaptor-like heterotetrameric complex. However, p47 is not enriched in preparations of clathrin-coated vesicles. Recruitment of the p47-containing complex onto cell membranes is stimulated by GTP gamma S and blocked by brefeldin A, indicating that, like other coat proteins, its membrane association is regulated by an ARF. The newly recruited complex is localized to non-clathrin-coated buds and vesicles associated with the TGN. Endogenous complex in primary cultures of neuronal cells is also localized to the TGN, and in addition, some complex is associated with the plasma membrane. These results indicate that the complex is a component of a novel type of coat that facilitates the budding of vesicles from the TGN, possibly for transporting newly synthesized proteins to the plasma membrane.

Characterization of a fourth adaptor-related protein complex.

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (epsilon, beta4, mu4, and sigma4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to approximately 10-20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The mu4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion.

Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

Tumor necrosis factor-alpha induces apoptosis of human adipose cells.

Tumor necrosis factor-alpha (TNF-alpha) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-alpha mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-alpha has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-alpha in limiting increase in fat mass. The aim of the present study was to determine if TNF-alpha could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-alpha at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-alpha induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-alpha induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-alpha in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-alpha.

Cytotrophoblast cells: a barrier to maternofetal transmission of passive immunity.

The human fetus receives passive immunity via the chorioallantoic placenta in the form of maternal immunoglobulin G (IgG) class antibodies. This provides protection against pathogens at a time when the fetus is immunologically naive. We localized endogenous human IgG using confocal laser scanning fluorescence microscopy and immunoelectron microscopy of frozen sections of chorionic villi from early and late gestation. With confocal microscopy we also investigated the distribution of a receptor for IgG (Fc gamma RIII; CD16) that is typically expressed on the surface of human leukocytes. Endogenous IgG was present in the syncytiotrophoblast that surrounds chorionic villi but underlying cytotrophoblast cells were devoid of endogenous antibody. Fc gamma RIII immunoreactivity was confined to the syncytiotrophoblast and was also absent from cytotrophoblast cells. We propose that cytotrophoblast cells present a barrier to the transmission of maternally derived IgG across the human placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast is known to express receptors for IgG. Cytotrophoblast cells form an almost complete epithelial layer underlying the syncytiotrophoblast at this stage of gestation, but this becomes discontinuous as the placenta matures, thus removing the cellular impediment to IgG transmission.

Heterogeneity of Fc gamma receptor-bearing cells in human term amniochorion.

Confocal microscopy has been used to study immunoreactivity to monoclonal antibodies directed against the three characterized human leukocyte Fc gamma receptors in term amniochorion. Fc gamma RI (CD64), Fc gamma RII (CDw32) and Fc gamma RIII (CD16) are expressed by cells of the maternal decidual layer associated with the amniochorion. These three subtypes of receptor are also expressed by cells of the fibroblast and reticular layers although not all cells of these layers express receptors which suggest that they are heterogeneous cell populations. We were not able to demonstrate Fc gamma RII immunoreactivity in decidual layer endothelial cells. Human IgG specific immunofluorescence was detected in the extracellular matrix of the tissue and associated with the Fc gamma receptor-bearing cells of mesenchymal origin and the maternal decidua, as demonstrated by dual labelling immunofluorescence studies. Two epithelial layers of cells (amniotic and trophoblast) lack Fc gamma receptors that are present in placental chorionic villi, and which may be necessary for transepithelial transport of IgG. The cells of these layers also contain no demonstrable endogenous IgG. These data support the hypothesis that maternally derived IgG is acquired by the fetus across the vascular system of the placenta only and not via a transamniochorion route. The demonstration of Fc gamma receptors on cells of extraembryonic mesodermal lineage is a novel finding that may suggest a potential immunological role within the amniochorion.

Targeting of a phogrin-green fluorescent protein chimaera to insulin secretory granules of pancreatic beta-cells in transgenic mice.

We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent protein (EGFP) targeted to pancreatic beta-cells by the rat insulin II promoter. The transgenic animals appear healthy, have normal weight gain and normal glucose tolerance. Morphological analyses of adult transgenic mice revealed that the fluorescent reporter molecule was specifically expressed in beta-cells of the pancreatic islet and not in extra-pancreatic tissues. The distribution of phogrin-EGFP in beta-cells, however, was heterogeneous with three distinct populations distinguishable by FACS analysis and immunofluorescence microscopy. Superficially-localized islets in the whole pancreas were readily visualized in the animals as was the developing endocrine pancreas in undissected 15.5 day old mouse embryos. We envisage that the animal will be an important resource for future investigations of islet development, regeneration and the molecular cell biology of insulin secretion.

Tumor necrosis factor-induced cytotoxicity is not related to rates of mitochondrial morphological abnormalities or autophagy-changes that can be mediated by TNFR-I or TNFR-II.

Tumor necrosis factor (TNF) may cause apoptosis or necrosis and induces mitochondrial changes that have been proposed to be central to cytotoxicity. We report similar patterns of TNF-induced mitochondrial morphological alterations and autophagy in cell types with differing sensitivity to TNF-induced cytotoxicity. Specific ligation of TNFR-I or TNFR-II induces different rates of apoptosis and mitochondrial morphological change, but similar rates of autophagy. These changes do not invariably lead to cell death, and survival or progression to apoptosis or necrosis following TNF exposure may depend in part on the extent of mitochondrial damage and/or the autophagic capacity of the cell.

Type VII collagen associated with the basement membrane of amniotic epithelium forms giant anchoring rivets which penetrate a massive lamina reticularis.

In human amnion a simple cuboidal epithelium and underlying fibroblast layer are separated by an almost acellular compact layer rich in collagen types I and III. This (>10 µm) layer, which may be a thick lamina reticularis, apparently presents an unusual set of conditions. Integration of the multilaminous tissue across it is apparently achieved by waisted structures which we have observed with the light microscope in frozen, paraffin-wax and semi-thin resin sections. We have also captured transmission and scanning electron micrographs of the structures. These structures which cross the compact layer we call "rivets". The composition of these "rivets" has been examined immunocytochemically and in three dimensions using the confocal laser scanning epi-fluorescence microscope. The rivets contain type VII collagen and an α6 integrin. They associate with type IV collagen containing structures (basement membrane lamina densa and spongy coils) and a special population of fibroblasts which may generate, maintain or anchor rivets to the underlying mesenchymal layer. Although type VII collagen is well known to anchor basal lamina to underlying mesodermal collagen fibres these "rivets" are an order of magnitude larger than any previously described type VII collagen containing anchoring structures. Intriguing possible functions of these features include nodes for growth of fibrous collagen sheets and sites of possible enzymatic degradation during regulated amnion weakening approaching term. If these sites are confirmed to be involved in amnion degradation and growth they may represent important targets for therapeutic agents that are designed to delay preterm premature rupture of the membranes a major cause of fetal morbidity and mortality.

The role of calcium and other ions in sorting and delivery in the late endocytic pathway.

The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain many luminal vesicles and are often referred to as MVBs (multivesicular bodies). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. The luminal ionic composition of organelles in the endocytic pathway is of considerable importance both in the trafficking of endocytosed ligands and in the membrane fusion events. In particular, H(+) ions play a role in sorting processes and providing an appropriate environment for the action of lysosomal acid hydrolases. Na(+)/H(+) exchangers in the endosomal membrane have been implicated in the formation of MVBs and sorting into luminal vesicles. Ca(2+) ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca(2+) in traffic through the late endocytic pathway, mutations in the gene encoding mucolipin-1, a lysosomal non-specific cation channel, result in abnormalities in lipid traffic and are associated with the autosomal recessive lysosomal storage disease MLIV (mucolipidosis type IV).

Ultrastructural distribution of endogenous immunoglobulin-G in human term amniochorion.

Maternal immunoglobulin-G (IgG) is known to be transported across the placental syncytiotrophoblast during the period when the human fetus is incapable of manufacturing these defensive molecules. In this study we investigated the possible role of the amniochorion, that surrounds the amniotic cavity in which the fetus lies, in the transfer of immunoglobulin. Endogenous IgG was localised in the amniochorion by confocal immunofluorescence microscopy and by ultrastructural labelling of ultrathin frozen tissue sections using the protein A-gold technique. Immunoreactivity was identified in the extracellular matrix tissues and necrotic amniotic epithelial cells. Healthy amniotic epithelial cells and cytotrophoblast cells of the chorion laeve were devoid of endogenous IgG. These results suggest a possible non-specific paracellular transport pathway between cytotrophoblast cells, which may conceivably contribute to the acquisition of passive immunity by the fetus, and offer a rational explanation for the presence of small quantities of maternal IgG in the amniotic fluid.

Ontogeny and distribution of Fc gamma receptors in the human placenta. Transport or immune surveillance?

The human fetus acquires maternal IgG via the chorioallantoic placenta. Utilising antibodies against 3 characterised subtypes of IgG receptor (Fc gamma R) expressed by human leucocytes, we show by confocal immunofluorescence microscopy that these molecules are also expressed by cells of the placenta. Fc gamma RI (CD64) is expressed by undifferentiated mesenchymal or fibroblast cells of 1st trimester and term chorionic villi. Punctate immunoreactivity for Fc gamma RII (CDw32) is found on capillary endothelial cells of term and 1st trimester villi. Fc gamma RIII (CD16) expression is observed in the trophoblast surrounding chorionic villi that forms the functional 'barrier' between mother and fetus. In 1st trimester villi this receptor is associated with a population of marginated vesicular inclusions of the syncytiotrophoblast. In term villi the receptor is concentrated in the apex of the syncytiotrophoblast, suggesting a possible role in the maternofetal transmission of passive immunity. All 3 subtypes of receptor are expressed by Hofbauer cells. We have been unable to demonstrate these receptors in cytotrophoblast cells. Results obtained using immunofluorescence and immunoelectron microscopic detection of endogenous IgG are consistent with the hypothesis that IgG is internalised into clathrin-coated pits and vesicles. Endogenous IgG was not demonstrable in cytotrophoblast cells. The significance of our inability to demonstrate IgG or specific receptor molecules for IgG in cytotrophoblast cells, and possible roles of Fc gamma receptor-bearing cells of the placenta are discussed.

The effects of cytochalasin D and phorbol myristate acetate on the apical endocytosis of ricin in polarised Caco-2 cells.

Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited > 95% by hypertonic and/or acid media, consistent with the major uptake route being clathrin-mediated. The presence of apical cell surface bound ricin-gold in clathrin coated pits and vesicles was observed by electron microscopy. An electron microscopic investigation in which ricin-gold bound to the apical surface was quantitated, showed that cytochalasin D, which inhibits apical but not basolateral endocytosis, prevented movement of ricin-gold along the microvillar surface. This was consistent with an actin bound mechanochemical motor within microvilli driving the movement of membranous components towards the cell body. Cytochalasin D also caused an increase in the number of coated pits observed at the apical cell surface relative to the number observed in untreated cells. Stimulation of apical endocytosis of ricin by phorbol 12-myristate 13-acetate showed the characteristics of being mediated by protein kinase C, was not due to an effect on ricin movement along the microvillar surface, and may be explained by increases in formation and pinching off of clathrin coated pits at the apical cell surface.

Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles.

Electron microscopy was used to evaluate the function and formation of dense core lysosomes. Lysosomes were preloaded with bovine serum albumin (BSA)-gold conjugates by fluid phase endocytosis using a pulse-chase protocol. The gold particles present in dense core lysosomes and late endosomes were flocculated, consistent with proteolytic degradation of the BSA. A second pulse of BSA-gold also accumulated in the pre-loaded dense core lysosomes at 37 degrees C, but accumulation was reversibly blocked by incubation at 20 degrees C. Time course experiments indicated that mixing of the two BSA-gold conjugates initially occurred upon fusion of mannose 6-phosphate receptor-positive/lysosomal glycoprotein-positive late endosomes with dense core lysosomes. Treatment for 5 hours with wortmannin, a phosphatidyl inositide 3-kinase inhibitor, caused a reduction in number of dense core lysosomes preloaded with BSA-gold and prevented a second pulse of BSA-gold accumulating in them. After wortmannin treatment the two BSA-gold conjugates were mixed in swollen late endosomal structures. Incubation of NRK cells with 0.03 M sucrose resulted in the formation of swollen sucrosomes which were morphologically distinct from preloaded dense core lysosomes and were identified as late endosomes and hybrid endosome-lysosome structures. Subsequent endocytosis of invertase resulted in digestion of the sucrose and re-formation of dense core lysosomes. These observations suggest that dense core lysosomes are biologically active storage granules of lysosomal proteases which can fuse with late endosomes and be re-formed from the resultant hybrid organelles prior to subsequent cycles of fusion and re-formation.

The effect of wortmannin on the localisation of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway.

Addition of wortmannin to normal rat kidney cells caused a redistribution of the lysosomal type I integral membrane proteins Igp110 and Igp120 to a swollen vacuolar compartment. This compartment did not contain the cation independent mannose 6-phosphate receptor and was depleted in acid hydrolases. It was distinct from another swollen vacuolar compartment containing the cation independent mannose 6-phosphate receptor. The swollen Igp110-positive compartment was accessible to a monoclonal antibody against Igp120 added extracellularly, showing that it had the characteristics of an endosomal compartment. Wortmannin had no gross morphological effect on the trans-Golgi network or lysosomes nor any effect on the delivery to the trans-Golgi network of endocytosed antibodies against the type I membrane protein TGN38. We propose that the observed effects of wortmannin were due to inhibition of membrane traffic between cation independent mannose 6-phosphate receptor-positive late endosomes and the trans-Golgi network and to inhibition of membrane traffic between a novel Igp120-positive, cation independent mannose 6-phosphate receptor-negative late endosomal compartment and lysosomes. The effects of wortmannin suggest a function for a phosphatidylinositol 3-kinase(s) in regulating membrane traffic in the late endocytic pathway.

The relationship between lumenal and limiting membranes in swollen late endocytic compartments formed after wortmannin treatment or sucrose accumulation.

Immunofluorescence and electron microscopy were used to evaluate the formation of swollen endosomes in NRK cells after treatment with wortmannin or sucrose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen late endocytic vacuoles, positive for lysosomal glycoproteins or cation-independent mannose 6-phosphate receptors, but those induced by wortmannin were characterised by time-dependent accumulation of lumenal vesicles, whereas those induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic acid, remained unchanged and was present within the lumen of the swollen vacuoles. Consumption of plasma membrane and peripheral early endosomes, and the appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is sufficient to account for the observed phenotype of cells treated with wortmannin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an explanation for the paucity of lumenal vesicles in swollen sucrosomes. Our data suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with the failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis.