Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa S3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0 degrees C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37 degrees C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits.     

Perforated MDCK cells support intracellular transport.

We have developed a method for perforating the plasma membrane of MDCK cells while retaining cellular functions. A nitrocellulose acetate filter was applied to the apical side of cells, grown on a glass coverslip, and allowed to dry. Segments of the apical plasma membrane adhered to the filter and were detached from the cell layer by shearing when the filter was peeled off. This allowed macromolecules such as antibodies and enzymes to diffuse into the cells. The cells were otherwise intact as judged by light and electron microscopy. The perforated cells maintained their capacity to support vesicular transport of proteins and lipids. Vesicular stomatitis virus infected cells readily incorporated [35S]methionine into G protein following permeabilization. This G protein was core-glycosylated during assembly in the endoplasmic reticulum, and was further transported to the trans Golgi with high efficiency. Experiments using lipid probes demonstrated that newly synthesized fluorescent sphingolipids were transported from the Golgi complex to the basolateral cell surface in perforated cells. Our results show that perforated cells provide a convenient and efficient alternative to cell-free assays for studying the molecular mechanism of intracellular transport.     

Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium

The toxic plant protein ricin binds to both the apical and basolateral surface domains of MDCK (strain I) cells grown on polycarbonate filters. Endocytosis of 125I-labeled ricin was not only higher from the basolateral than from the apical surface--an observation which can be explained by the higher surface area of the basolateral surface--but it also appeared to be more efficient when measured as a percentage of total cell-associated ricin. Monovalent ricin-horseradish peroxidase (Ri-HRP), which is known to behave like native ricin with respect to intracellular transport, also binds to, and is taken up from, both the apical and the basolateral surfaces. Initially, after 10 to 15 min, molecules taken up from the two surface domains at 37 degrees C are present in two separate (basolateral and apical) early endosomal populations. This can also be obtained by incubating for 60 min at 18 degrees C. However, after 30 to 60 min at 37 degrees C, most internalized ligand is found in apical lysosomes, regardless from which surface endocytosis took place. Experiments with endocytosis of cationized ferritin from the apical pole and HRP or Ri-HRP from the basolateral pole showed that intermixing in apical lysosomes (or prelysosomes) of molecules taken up from the two poles occurs. Bidirectional transcytosis involving coated pits of both 125I-labeled ricin and Ri-HRP was demonstrated and was found to be most efficient (as measured in per cent of endocytosed toxin) from the apical pole. Transcytosis was strongly reduced at 18 degrees C, and no transepithelial transport of ricin could be measured at 4 degrees C. Transcytosed ricin was intact and could intoxicate new cells. Finally, delivery of ricin internalized from both the apical and the basolateral surface to the apically localized trans-Golgi network occurred at 37 degrees C but not at 18 degrees C, and ricin inhibited protein synthesis largely with the same kinetics following uptake from the two poles. Incubation at 18 degrees C strongly inhibited the toxic effect of ricin. These data show that ricin can intoxicate epithelia from both sides and also penetrate tight epithelial barriers in intact form.     

Endocytosis in polarized cells.

Recent biochemical and morphological studies using the MDCK cell line have provided insights into the organization of the endocytic pathways in an epithelial cell. The cytoskeletal organization of these cells has been described and evidence for the involvement of microtubules in facilitating endocytic traffic has been obtained. The findings with this model system are compared to results from in vivo studies of the endocytic pathways from the surface domains of specialized epithelial cells and to studies of endocytosis in neurons.     

Involvement of VIP36 in intracellular transport and secretion of glycoproteins in polarized Madin-Darby canine kidney (MDCK) cells

VIP36, an intracellular lectin that recognizes high mannose-type glycans (Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999) Glycobiology 9, 833-839), was shown to localize not only to the early secretory pathway but also to the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In the plasma membrane, VIP36 exhibited an apical-predominant distribution, the apical/basolateral ratio being approximately 2. Like VIP36, plasma membrane glycoproteins recognized by VIP36 were found in the apical and basolateral membranes in the ratio of approximately 2 to 1. In addition, secretory glycoproteins recognized by VIP36 were secreted approximately 2-fold more efficiently from the apical membrane than from the basolateral membrane. Thus, the apical/basolateral ratio of the transport of VIP36-recognized glycoproteins was correlated with that of VIP36 in MDCK cells. Upon overproduction of VIP36 in MDCK cells, the apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins were changed from approximately 2 to approximately 4, and the secretion of VIP36-recognized glycoproteins was greatly stimulated. In contrast to the overproduction of VIP36, that of a mutant version of VIP36, which has no lectin activity, was of no effect on the distribution of glycoproteins to apical and basolateral membranes and inhibited the secretion of VIP36-recognized glycoproteins. Furthermore, the overproduction of VIP36 greatly stimulated the secretion of a major apical secretory glycoprotein of MDCK cells, clusterin, which was found to carry at least one high mannose-type glycan and to be recognized by VIP36. In contrast to the secretion of clusterin, that of a non-glycosylated apical-secretion protein, galectin-3, was not stimulated through the overproduction of VIP36. These results indicated that VIP36 was involved in the transport and sorting of glycoproteins carrying high mannose-type glycan(s).     

FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin-Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.     

Clostridium perfringens epsilon toxin rapidly decreases membrane barrier permeability of polarized MDCK cells

Epsilon toxin is produced by Clostridium perfringens types B and D which are responsible for fatal intestinal diseases in animals. The main biological activity of epsilon toxin is the production of oedema in various organs. We have previously found that epsilon toxin forms a large membrane complex in MDCK cells which is not internalized into cell, and induces cell volume enlargement and loss of cell viability (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., Popoff, M. R. (1997) J Bacteriol 179, 6480-6487). Here, we show that epsilon toxin is very potent to decrease the trans-epithelial electrical resistance of polarized MDCK cells grown on filters without altering the organization of the junctional complexes. The dose-dependent decrease in trans-epithelial electrical resistance, more marked when the toxin was applied to the apical side than to the basal side of MDCK cells, was associated with a moderate increase of the paracellular permeability to low-molecular-weight compounds but not to macromolecules. Epsilon toxin probably acts by forming large membrane pores which permit the flux of ions and other molecules such as the entry of propidium iodide and finally to the loss of cell viability.     

Effect of monensin on ricin and fluid phase transport in polarized MDCK cells.

The effect of monensin on endocytosis, transcytosis, recycling and transport to the Golgi apparatus in filter-grown Madin-Darby canine kidney (MDCK) cells was investigated using 125I-labeled ricin as a marker for membrane transport, and horseradish peroxidase (HRP) as a marker for fluid phase transport. Monensin (10 microM) stimulated transcytosis of both markers about 3-fold in the basolateral to apical direction. Transcytosis of HRP in the opposite direction, apical to basolateral, was reduced to approximately 50% of the control by monensin, whereas that of ricin was slightly increased. Recycling of markers endocytosed from the apical surface was reduced in the presence of monensin and there was an increased accumulation of both ricin and HRP in the cells. Transport of ricin to the Golgi apparatus increased to the same extent as the increase in intracellular accumulation. No change in recycling or accumulation was observed with monensin when the markers were added basolaterally, but transport of ricin to the Golgi apparatus increased almost 3-fold. Our results indicate that basolateral to apical transcytosis is increased in the absence of low endosomal pH, and they suggest that apical to basolateral transcytosis of a membrane-bound marker (ricin) is affected by monensin differently from that of a fluid phase marker (HRP).     

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.     

SNARE protein trafficking in polarized MDCK cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N-ethyl maleimide-sensitive factor attachment protein receptors (SNARE) proteins of vesicle-associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin–Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.     

Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells

We have studied the effects of brefeldin A (BFA) on endocytosis and intracellular traffic in polarized MDCK cells by using the galactose-binding protein toxin ricin as a membrane marker and HRP as a marker of fluid phase transport. We found that BFA treatment rapidly increased apical endocytosis of both ricin and HRP, whereas basolateral endocytosis was unaffected, as was endocytosis of HRP in the poorly polarized carcinoma cell lines HEp-2 and T47D. Tubular endosomes were induced by BFA both apically and basolaterally in some MDCK cells, comparable with those seen in HEp-2 and T47D cells. In addition, in MDCK cells, BFA induced formation of small (< 300 nm) vesicles, labeled both after apical and basolateral uptake of HRP, as well as some very large (> 700 nm) vacuoles, which were only labeled when HRP was present in the apical medium. In contrast, neither in MDCK nor in HEp-2 or T47D cells, did BFA have any effect on lysosomal morphology. Moreover, transcytosis in the basolateral-apical direction was stimulated both for HRP and ricin. Other vesicular transport routes were less affected or unaffected by BFA treatment. Two closely related structural analogues of BFA (B16 and B21), unable to produce the changes in Golgi and endosomal morphology seen after BFA treatment in a number of different cell lines, were also unable to mimic the effects of BFA on MDCK cells.     

The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport,intracellular vectorization,and more

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.     

Endocytosis and phosphate transport in OK epithelial cells

Endocytosis was studied in OK epithelial cells, an established cell line from opossum kidney. The presence of fluid-phase endocytosis in these cells was demonstrated by measuring cell uptake of lucifer yellow and horseradish peroxidase. The intracellular distribution of lucifer yellow fluorescence was consistent with uptake by endocytosis. Endocytosis was inhibited in medium made hyperosmolar by addition of sucrose. In hyperosmolar medium the action of parathyroid hormone on Na+/phosphate cotransport was significantly diminished. We suggest that an intact endocytic mechanism is required for the full inhibitory effect of parathyroid hormone on Na+/phosphate cotransport.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

N-glycans mediate apical recycling of the sialomucin endolyn in polarized MDCK cells

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N-glycan-dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin-Darby canine kidney cells. Terminal processing of a subset of endolyn's N-glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine-based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N-glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.     

Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.     

Impact and intracellular localization of antibodies to DNA in MDCK cells

A study was performed with liquid chromatography of highly purified antibodies to DNA of IgG from blood serum of healthy donors-SLE or RA patients and their clinically healthy relatives. It was shown that, depending on the pathology, these antibodies were distinguished by DNA—hydrolyzing activity. These antibodies were incubated with MDCK cells. It was found that they were mostly localized in the vicinity of the nucleus and affected the cellular morphology, number of viable cells, and chromatin of MDCK cells differently. The biological role of antibodies to DNA is discussed.