Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.     

Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins.     

Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane

Generation of intestinal epithelial lipid polarity was studied in Caco-2 cells. Confluent monolayers on filters incorporated the exchangeable lipid N-6-NBD-aminocaproyl-sphingosine (C6-NBD-ceramide) from liposomes. The fluorescent ceramide was converted equally to C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, analogues of lipids enriched on the apical and basolateral surface, respectively, of intestinal cells in vivo. Below 16 degrees C, where vesicular traffic is essentially blocked, each fluorescent product accumulated in the Golgi area. At 37 degrees C, 50% had been transported to the cell surface within 0.5 h, as measured by selective extraction of the fluorescent lipids onto BSA in the medium ("back-exchange") at 10 degrees C. Transport to the two surfaces could be assayed separately, as a diffusion barrier existed for both NBD-lipids and BSA. C6-NBD-glucosylceramide was enriched twofold apically, whereas C6-NBD-sphingomyelin was equally distributed over both domains. Polarities did not decrease when 37 degrees C incubations were carried out in the presence of increasing BSA concentrations to trap the fluorescent lipids immediately after their arrival at the cell surface. Within 10 min from the start of synthesis, both products displayed their typical surface polarity. Lipid transcytosis displayed a half time of hours. In conclusion, newly synthesized sphingolipids in Caco-2 cells are sorted before reaching the cell surface. Transcytosis is not required for generating the in vivo lipid polarity.     

Fluorescent analogues of plasma membrane sphingolipids are sorted to different intracellular compartments in astrocytes; Harmful effects of chronic ethanol exposure on sphingolipid trafficking and metabolism

Sphingolipids are basic constituents of cellular membranes and are essential for numerous functions such as intracellular signalling. They are transported along the exocytic and endocytic pathways in eukaryotic cells. After endocytosis, fluorescent-labelled sphingolipids are sorted to distinct intracellular organelles prior to recycling (via early/recycling endosomes) or degradation (late endosomes/lysosomes). Here we examine, in primary cultures of rat astrocytes, the internalisation routes followed by C(6)-NBD-glucosylceramide (NBD-GlcCer) and C(6)-NBD-sphingomyelin (NBD-SM) and the effects of ethanol on their endocytic trafficking. Endocytosed plasma membrane NBD-GlcCer and NBD-SM are diverted to the Golgi apparatus and lysosomes, respectively. These different internalisation pathways are maintained regardless of the differentiation stage of astrocytes. Chronic ethanol exposure did not alter this endocytic sorting, but delayed the internalisation of both NBD-sphingolipids. Moreover, ethanol also stimulated the in situ metabolism of NBD-ceramide to NBD-GlcCer and NBD-SM. We conclude that in rat astrocytes internalised plasma membrane NBD-sphingolipids are sorted to different subcellular compartments. The exposure to chronic ethanol perturbed the lipid endocytic process and stimulated the de novo synthesis of NBD-sphingolipids, shifting the balance of sphingolipid metabolism in favour of the sphingomyelin pathway.     

Endocytosis of NBD-sphingolipids in neurons: exclusion from degradative compartments and transport to the Golgi complex

Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non-neuronal cultured cell lines, plasma membrane short-chain sphingomyelin and glucosylceramide are recycled via endosomes or sorted to degradative compartments. However, depending on cell type and lipid membrane composition, short-chain glucosylceramide can also be diverted to the Golgi complex. Here, we show that NBD-labeled glucosylceramide and sphingomyelin are transported from the plasma membrane to the Golgi complex in cultured rat hippocampal neurons irrespective of the stage of neuronal differentiation. Golgi complex localization was confirmed by colocalization and Golgi disruption studies, and importantly did not result from conversion of NBD-glucosylceramide or NBD-sphingomyelin to NBD-ceramide. Double-labeling experiments with transferrin or wheat-germ agglutinin showed that NBD-sphingolipids are first internalized to early/recycling endosomes, and subsequently transported to the Golgi complex. The internalization of these two sphingolipid analogs was energy and temperature dependent, and their intracellular transport was insensitive to the NBD fluorescence quencher sodium dithionite. These results indicate that vesicles mediate the transport of internalized NBD-glucosylceramide and NBD-sphingomyelin to the Golgi complex.     

Trafficking of Acetyl‐C16‐Ceramide‐NBD with Long‐Term Stability and No Cytotoxicity into the Golgi Complex

The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐labeled C6‐ceramide (NBD‐C6‐ceramide, a ceramide having an NBD‐bound C6‐N‐acyl chain) and Golgi‐specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl‐C16‐ceramide‐NBD (a ceramide having an acetylated C‐1 hydroxyl group, C16‐N‐acyl chain, and NBD‐bound C15‐sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl‐C16‐ceramide‐NBD were investigated. Acetyl‐C16‐ceramide‐NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD‐C6‐ceramide, acetyl‐C16‐ceramide‐NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl‐C16‐ceramide‐NBD‐labeled cells before and after mitosis.      

Distribution and metabolism of fluorescent sphingosines and corresponding ceramides bearing the diphenylhexatrienyl (DPH) fluorophore in cultured human fibroblasts.

Fluorescent D-erythro-sphingosines bearing the diphenyl-1,3,5-hexatrienyl group (DPH) as fluorophore were synthesized for the first time. Two isomers, the DPH-4(E)- and DPH-4(Z)-sphingosine [(2S,3R)-2-amino-6-(p-(18-phenyl)-13,15,17(E,E,E)-hexatrienyl)phenylh ex- 4(E/Z)-en-1,3-diol], and the N-hexanoyl derivative of DPH-4(E)-sphingosine (C6-DPH-ceramide) were studied for their distribution and metabolism in cultured human skin fibroblasts. Both DPH-sphingosines (4-trans and 4-cis) were not significantly acylated to ceramide in living cells, but converted to ceramide in vitro by microsomal protein from mouse brain, although slower than natural D-erythro-sphingosine. DPH-4(Z)-sphingosine showed the same Km like D-erythro-sphingosine (155 microM), but had a lower Vmax value, 0.85 instead of 1.9 nmol/mgh. An even poorer substrate was DPH-4(E)-sphingosine with a Km of 220 microM and a Vmax of 0.81 nmol/mgh. In cultured human fibroblasts, C6-DPH-ceramide was rapidly anabolized mainly to sphingomyelin. In addition, small quantities of glucosylceramide were also formed. DPH-sphingosines were easily incorporated into plasma membranes of cultured fibroblasts and are likely to undergo flip flop since intracellular membranes also became labeled, when endocytosis was blocked at low temperature (7 degrees C). The N-hexanoyl-DPH-trans-sphingosine, C6-DPH-ceramide, like NBD-C6-ceramide (Lipsky, N. G., R. E. Pagano: Science 228, 745-747 (1985)) labeled intracellular membranes at 7 degrees C and predominantly Golgi membranes at 37 degrees C. Like NBD-C6-ceramide (Pagano, R. E., M. A. Sepanski, O. C. Martin: J. Cell Biol. 109, 2067-2079 (1989)) the C6-DPH-ceramide also stained the Golgi complex in prefixed cells whereas DPH-trans- and DPH-cis-sphingosine did not, indicating that it is the ceramide structure rather than the fluorophore itself which is responsible for this staining. DPH-sphingosine opens a way for chemical synthesis of DPH-glycolipids and DPH-sphingomyelin which would well serve as donors in fluorescence energy transfer experiments to study possible sphingolipid clustering in biological membranes.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Internalization and trafficking of neurotensin via NTS3 receptors in HT29 cells

The neurotensin receptor-3, originally identified as sortilin, is unique among neuropeptide receptors in that it is a single trans-membrane domain, type I receptor. To gain insight into the functionality of neurotensin receptor-3, we examined the neurotensin-induced intracellular trafficking of this receptor in the human carcinoma cell line HT29, which expresses both neurotensin receptor-1 and -3 sub-types. At steady state, neurotensin receptor-3 was found by sub-cellular fractionation and electron microscopic techniques to be predominantly associated with intracellular elements. A small proportion (approximately 10%) was associated with the plasma membrane, but a significant amount (approximately 25%) was observed inside the nucleus. Following stimulation with neurotensin (NT), neurotensin/neurotensin receptor-3 complexes were internalized via the endosomal pathway. This internalization entailed no detectable loss of cell surface receptors, suggesting compensation through either recycling or intracellular receptor recruitment mechanisms. Internalized ligand and receptors were both sorted to the pericentriolar recycling endosome/Trans-Golgi Network (TGN), indicating that internalized neurotensin is sorted to this compartment via neurotensin receptor-3. Furthermore, within the Trans-Golgi Network, neurotensin was bound to a lower molecular form of the receptor than at the cell surface or in early endosomes, suggesting that signaling and transport functions of neurotensin receptor-3 may be mediated through different molecular forms of the protein. In conclusion, the present work suggests that the neurotensin receptor-3 exists in two distinct forms in HT29 cells: a high molecular weight, membrane-associated form responsible for neurotensin endocytosis from the cell surface and a lower molecular weight, intracellular form responsible for the sorting of internalized neurotensin to the Trans-Golgi Network.     

Endocytosis of mannose-6-phosphate binding sites by mouse T-lymphoma cells

The endocytosis and intracellular transport of mannose-6-phosphate conjugated to bovine serum albumin (Man-6-P:BSA) by mouse T-lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man-6-P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man-6-P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent- or 125I-labeled Man-6-P:BSA at 0 degree C revealed a uniform distribution of the Man-6-P binding sites over the cell surface. Competition experiments indicate that the Man-6-P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37 degrees C, endocytosis of Man-6-P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5-15-min incubation of cells at 37 degrees C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man-6-P:BSA in the Golgi region after 15-30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man-6-P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30-min incubation at 37 degrees C, the internalized Man-6-P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co-stained for acid phosphatase. These results demonstrate a temporal participation of clathrin-containing coated vesicles during the initial endocytosis of Man-6-P binding sites and that one step in the Man-6-P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.     

Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner

Ebolavirus (EBOV) is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with mortality rates of up to 90% in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolae-mediated endocytosis in EBOV entry; however, ebolavirus virions are long, filamentous particles that are larger than the plasma membrane invaginations that characterize clathrin- or caveolae-mediated endocytosis. The mechanism of EBOV entry remains, therefore, poorly understood. To better understand Ebolavirus entry, we carried out internalization studies with fluorescently labeled, biologically contained Ebolavirus and Ebolavirus-like particles (Ebola VLPs), both of which resemble authentic Ebolavirus in their morphology. We examined the mechanism of Ebolavirus internalization by real-time analysis of these fluorescently labeled Ebolavirus particles and found that their internalization was independent of clathrin- or caveolae-mediated endocytosis, but that they co-localized with sorting nexin (SNX) 5, a marker of macropinocytosis-specific endosomes (macropinosomes). Moreover, the internalization of Ebolavirus virions accelerated the uptake of a macropinocytosis-specific cargo, was associated with plasma membrane ruffling, and was dependent on cellular GTPases and kinases involved in macropinocytosis. A pseudotyped vesicular stomatitis virus possessing the Ebolavirus glycoprotein (GP) also co-localized with SNX5 and its internalization and infectivity were affected by macropinocytosis inhibitors. Taken together, our data suggest that Ebolavirus is internalized into cells by stimulating macropinocytosis in a GP-dependent manner. These findings provide new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection.     

Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms

We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]- glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB- galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB- sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane.     

Mechanism of uptake of C105Y, a novel cell-penetrating peptide

C105Y, a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acid sequence corresponding to residues 359-374 of alpha1-antitrypsin, enhances gene expression from DNA nanoparticles. To investigate how this enhancement occurs, C105Y was fluorescently labeled to study its uptake and intracellular trafficking. When human hepatoma cells (HuH7) were incubated with fluorescently labeled C105Y for as little as 3 min, C105Y displayed nuclear and cytoplasmic staining with enrichment of fluorescent signal in the nucleus and nucleolus. Uptake and nucleolar localization were observed with the short sequence PFVYLI, but not with SIPPEVKFNK, and the D-isomer was readily taken up into cells but not into the nucleus. We found that the C105Y peptide is routed to the nucleolus very rapidly in an energy-dependent fashion, whereas membrane translocation and nuclear localization are energy-independent. When we tested the involvement of known endocytosis pathways in uptake and trafficking of this peptide, we demonstrated that C105Y peptide is internalized by a clathrin- and caveolin-independent pathway, although lipid raft-mediated endocytosis may play a role in peptide intracellular trafficking. Efficient energy-independent cell entry with rapid nuclear localization probably accounts for enhancement of gene expression from inclusion of C105Y into DNA nanoparticles.     

Selective caveolin-1-dependent endocytosis of glycosphingolipids

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.     

Regulation of constitutive TCR internalization by the zeta-chain

The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the zeta-chain in this process. We found that cell surface TCRs lacking zeta were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length zeta led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4(+)CD8(+) thymocytes from zeta-deficient mice, whereas TCR internalization on thymocytes from CD3-delta deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for zeta in the regulation of constitutive receptor internalization. Furthermore, chimeric zeta molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular zeta-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the zeta-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.     

A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway

The fluorescent phospholipid analog N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was inserted into the plasma membrane of Baby hamster kidney cells at low temperature (2 degrees C). The mobility characteristics of the analog--as revealed by fluorescence photobleaching recovery--were very similar to those of membrane-inserted 1-acyl-2[6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]caproyl] phosphatidylcholine (C6-NBD-PC). Upon warming to 37 degrees C, followed by a 1-h incubation, all N-Rh-PE was located intracellularly. By contrast, after the same time interval, approximately 10% of the cell-associated PC-derivative was found intracellularly. Furthermore, the analogs moved to different intracellular sites, as N-Rh-PE associates with perinuclear and peri-Golgi structures, whereas C6-NBD-PC appears mainly in the Golgi complex. Colocalization with organelle-specific probes and Percoll gradient analysis identified the N-Rh-PE-labeled structures as lysosomes. Temperature and energy-dependent experiments supported the endocytic pathway as the mechanism of N-Rh-PE internalization. The mechanism of N-Rh-PE internalization appears to differ from that of C6-NBD-PC. In conjunction with a difference in the efficiency of removal of the lipid derivatives from the plasma membrane, the results suggest that N-Rh-PE is selectively internalized, implying that sorting of the lipid analogs already occurs at the level of the plasma membrane. The distinct difference in physical appearance of the probes after membrane insertion, i.e., N-Rh-PE being present as small clusters and C6-NBD-PC as monomers, could explain the selective sorting and internalization of N-Rh-PE. The results demonstrate that N-Rh-PE may serve as a useful marker for studying membrane traffic during endocytosis.     

Endocytosis and trafficking of human lactoferrin in macrophage-like human THP-1 cells (1)

Different cell types have been reported to internalize lactoferrin (Lf) by specific or nonspecific receptors. Our studies focused on the endocytic pathway of human Lf in macrophage-like THP-1 cells. Lactoferrin was found to be internalized by THP-1 cells differentiated with phorbol myristate acetate. Incubation of cells with chlorpromazine and dansylcadaverine, inhibitors of clathrin-dependent endocytosis, led to a 50% inhibition of Lf internalization compared with untreated cells. Bafilomycin A1 and NH(4)Cl treatment also resulted in 40%-60% inhibition, respectively, suggesting that the internalization of Lf may partly be mediated by acidic endosome-like organelles. Endocytic uptake of Lf was also cholesterol-dependent, as shown by methyl-β-cyclodextrin or nystatin treatment of the cells prior to internalization. Partial colocalization of Lf and EEA-1, a marker specific for early endosomes, could be observed. Colocalization of Lf with a specific endoplasmic reticulum marker was also detected. Our results suggest that Lf is internalized mainly by the clathrin-dependent pathway in THP-1 cells and targets the ER. The physiological consequences of this intracellular trafficking will be the subject of future investigations.     

Functionally different pools of Shiga toxin receptor, globotriaosyl ceramide, in HeLa cells

Many studies have investigated the intracellular trafficking of Shiga toxin, but very little is known about the underlying dynamics of its cellular receptor, the glycosphingolipid globotriaosyl ceramide. In this study, we show that globotriaosyl ceramide is required not only for Shiga toxin binding to cells, but also for its intracellular trafficking. Shiga toxin induces globotriaosyl ceramide recruitment to detergent-resistant membranes, and subsequent internalization of the lipid. The globotriaosyl ceramide pool at the plasma membrane is then replenished from internal stores. Whereas endocytosis is not affected in the recovery condition, retrograde transport of Shiga toxin to the Golgi apparatus and the endoplasmic reticulum is strongly inhibited. This effect is specific, as cholera toxin trafficking on GM(1) and protein biosynthesis are not impaired. The differential behavior of both toxins is also paralleled by the selective loss of Shiga toxin association with detergent-resistant membranes in the recovery condition, and comparison of the molecular species composition of plasma membrane globotriaosyl ceramide indicates subtle changes in favor of unsaturated fatty acids. In conclusion, this study demonstrates the dynamic behavior of globotriaosyl ceramide at the plasma membrane and suggests that globotriaosyl ceramide-specific determinants, possibly its molecular species composition, are selectively required for efficient retrograde sorting on endosomes, but not for endocytosis.     

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.     

Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.