Complex gangliosides are apically sorted in polarized MDCK cells and internalized by clathrin-independent endocytosis Pilar M. Crespo, Natalia von Muhlinen, Ramiro Iglesias-Bartolome ´ and Jose L. Daniotti Centro de Investigaciones en Quı ´ mica Biolo ´ gica de Co ´ rdoba (CIQUIBIC, UNC-CONICET), Departamento de Quı ´ mica Biolo ´ gica, Facultad de Ciencias Quı ´ micas, Universidad Nacional de Co ´ rdoba, Argentina Gangliosides are complex glycosphingolipids contain- ing one or more sialic acid residues, which are mainly located at the outer leaflet of the plasma membrane of eukaryotic cells. They participate in cell surface events, such as the modulation of growth factor receptors and cell-to-cell and cell-to-matrix interactions [1–6]. The synthesis of gangliosides is carried out in the lumen of the Golgi complex by a complex system of membrane- bound glycolipid acceptors, glycosyltransferases and sugar nucleotide transporters [7,8]. After synthesis, gangliosides leave the Golgi complex via the lumenal surface of transport vesicles. In this context, we have Keywords gangliosides; glycolipids; intracellular trafficking; MDCK cells; polarized cells Correspondence J. L. Daniotti, Centro de Investigaciones en Quı ´ mica Biolo ´ gica de Co ´ rdoba (CIQUIBIC, UNC-CONICET), Departamento de Quı ´ mica Biolo ´ gica, Facultad de Ciencias Quı ´ micas, Universidad Nacional de Co ´ rdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA Co ´ rdoba, Argentina Fax: +54 351 433 4074 Tel: +54 351 433 4168 ⁄ 4171 E-mail: daniotti@dqb.fcq.unc.edu.ar (Received 4 August 2008, revised 21 September 2008, accepted 8 October 2008) doi:10.1111/j.1742-4658.2008.06732.x Gangliosides are glycosphingolipids mainly present at the outer leaflet of the plasma membrane of eukaryotic cells, where they participate in recogni- tion and signalling activities. The synthesis of gangliosides is carried out in the lumen of the Golgi apparatus by a complex system of glycosyltrans- ferases. After synthesis, gangliosides leave the Golgi apparatus via the lumenal surface of transport vesicles destined to the plasma membrane. In this study, we analysed the synthesis and membrane distribution of GD3 and GM1 gangliosides endogenously synthesized by Madin–Darby canine kidney (MDCK) cell lines genetically modified to express appropriate gan- glioside glycosyltransferases. Using biochemical techniques and confocal laser scanning microscopy analysis, we demonstrated that GD3 and GM1, after being synthesized at the Golgi apparatus, were transported and accu- mulated mainly at the plasma membrane of nonpolarized MDCK cell lines. More interestingly, both complex gangliosides were found to be enriched mainly at the apical domain when these cell lines were induced to polarize. In addition, we demonstrated that, after arrival at the plasma membrane, GD3 and GM1 gangliosides were endocytosed using a clathrin-independent pathway. Then, internalized GD3, in association with a specific monoclonal antibody, was accumulated in endosomal compartments and transported back to the plasma membrane. In contrast, endocytosed GM1, in associa- tion with cholera toxin, was transported to endosomal compartments en route to the Golgi apparatus. In conclusion, our results demonstrate that complex gangliosides are apically sorted in polarized MDCK cells, and that GD3 and GM1 gangliosides are internalized by clathrin-indepen- dent endocytosis to follow different intracellular destinations. Abbreviations CHO, Chinese hamster ovary; CTx, cholera toxin; DiI, 1,1¢-dioctadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate; Eps15, epidermal growth factor receptor pathway substrate clone 15; GalNAc-T, UDP-GalNAc:LacCer ⁄ GM3 ⁄ GD3 N-acetylgalactosaminyltransferase; Gal-T2, UDP-Gal:GA2 ⁄ GM2 ⁄ GD2 ⁄ GT2 galactosyltransferase; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; LacCer, lactosylceramide; MDCK, Madin–Darby canine kidney; PI, propidium iodide; Sial-T2, CMP-NeuAc:GM3 sialyltransferase; Tf, transferrin; TGN, trans-Golgi network; YFP, yellow fluorescent protein. FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS 6043 demonstrated, in Chinese hamster ovary (CHO)-K1 cells, that gangliosides traffic from the trans-Golgi network (TGN) to the plasma membrane by a Rab11- independent and Brefeldin A-insensitive exocytic pathway [9]. Gangliosides have been found to reside in glycosphingolipid-enriched microdomains (also called detergent-resistant membranes or rafts), dynamic assemblies of cholesterol, saturated phospholipids and sphingolipids [10–12]. The renal epithelial Madin–Darby canine kidney (MDCK) cell line is a recognized cellular model system for the study of protein targeting because of its ability, when grown to confluence, to form a polarized mono- layer [13]. Polarized epithelial cell surface membranes are divided into apical and basolateral domains possess- ing distinct protein and lipid compositions that are sepa- rated by tight junctions. It is known that proteins can be targeted directly to the apical or basolateral membrane from the TGN via the exocytic pathway [14–16]. They can also be targeted indirectly by being delivered to one domain, typically the basolateral domain, endocytosed and then redirected to the opposite domain in a process termed ‘transcytosis’ [17]. Alternatively, proteins can be randomly targeted to both domains and achieve their asymmetric distribution by selective stabilization at one plasma membrane [18]. In MDCK cells, newly synthe- sized apical and basolateral membrane proteins are seg- regated into separate transport vesicles within the TGN by virtue of sorting signals within the protein [13]. Spe- cifically, the apical targeting of proteins within MDCK cells can be mediated by sequestration into apical trans- port vesicles via association with sorting platforms rich in cholesterol and sphingolipid-rich lipid rafts at the level of the TGN [19,20]. The prevention of the associa- tion of apical proteins with lipid rafts perturbs the apical sorting of these proteins [21]. Polarized distribution of sphingolipids has been reported in different cell types. In migrating lympho- cytes, GM1 localizes to the uropods, whereas another form, GM3, segregates to the leading edge [22]. In fully polarized human hepatoma HepG2 cells, C6-NBD-GlcCer, a fluorescent sphingolipid analogue of glucosylceramide, recycles between the subapical compartment and the apical, bile canalicular membrane. By contrast, C6-NBD-SM, a fluorescent sphingolipid analogue of sphingomyelin, initially accumulates in the subapical compartment, but is ulti- mately transported to the basolateral membrane [23]. Apical membranes from MDCK cells have generally been found to be enriched mainly in neutral glycos- phingolipids and sphingomyelin, whereas phosphati- dylcholine is concentrated in the basolateral domain (for a discussion, see [24–26]). In this study, we analysed the subcellular distribu- tion of GD3 and GM1 gangliosides endogenously expressed in both polarized and nonpolarized MDCK cell lines stably transfected to express CMP-NeuAc: GM3 sialyltransferase (Sial-T2, GD3 synthase) and UDP-GalNAc:LacCer ⁄ GM3 ⁄ GD3 N-acetylgalactos- aminyltransferase (GalNAc-T, GM2 synthase) glyco- syltransferases. GD3 and GM1 gangliosides were found to be enriched mainly at the apical domain when these cell lines were induced to polarize. In addi- tion, we found that GD3 ganglioside, in association with a specific monoclonal antibody, was endocytosed using a clathrin-independent pathway and recycled back to the plasma membrane. GM1 ganglioside, in association with cholera toxin (CTx), was also actively endocytosed in MDCK cells and transported to the en- dosomal compartment en route to the Golgi complex. Results MDCK cells synthesize and express GD3 and GM1 gangliosides at the cell surface after transfection with Sial-T2 and GalNAc-T MDCK cell lines expressing different gangliosides were generated by stable transfection with the cDNA encod- ing either chicken full-length Sial-T2 [tagged with the hemagglutinin (HA) epitope] or human full-length Gal- NAc-T (tagged with the c-myc epitope) under the con- trol of constitutive promoters. The expression of the recombinant glycosyltransferases was characterized by double immunostaining. GalNAc-T and Sial-T2 were found to be located predominantly in a region near the cell nucleus, colocalizing with the area of immunostaining of GM130, a cis-medial Golgi marker [27] (Fig. 1A). Moreover, full-length Sial-T2 and Gal- NAc-T colocalized with their respective fluorescent truncated versions (Sial-T2-YFP and GalNAc-T-YFP; YFP, yellow fluorescent protein), which were also found to concentrate at the Golgi apparatus in another epithelial cell [28] (results not shown). Wild-type MDCK cells predominantly express the ganglioside GM3, as shown in the pattern of radioactive lipids metabolically labelled from d-[U- 14 C]galactose (Fig. 1B, MDCK wt). Cells stably transfected with the cDNA encoding chicken Sial-T2 (Clone 13) mostly synthesize GM3 and GD3 (MDCK Sial-T2); a minor amount of GT3 was also observed, probably also synthe- sized by Sial-T2, as reported previously [29]. The notice- able decrease in lactosylceramide (LacCer) labelling was most probably a result of conversion to GM3, which is transformed to GD3 and GT3 by the activity of trans- fected sialyltransferase. In contrast, MDCK cells stably Ganglioside trafficking in MDCK cells P. M. Crespo et al. 6044 FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS expressing the human full-length GalNAc-T cDNA (Clone 22) synthesize GM3 and GA2 (MDCK GalNAc- T). The expression of GM1 was below the limit of detec- tion, even at longer exposure times, probably indicating inefficient coupling between GalNAc-T and GM3, its endogenous substrate, and ⁄ or a very low activity of endogenous UDP-Gal:GA2 ⁄ GM2 ⁄ GD2 galactosyltrans- ferase (Gal-T2) in MDCK cells to catalyse the conversion of GM2 to GM1. In addition, no expression of GM1 was observed in an extract from GalNAc-T-expressing MDCK cells when analysed by TLC immunostaining using an antibody to GM1 (results not shown). A B C Fig. 1. Characterization of stably transfected MDCK cell clones. (A) MDCK clones stably expressing Sial-T2-HA (MDCK Clone 13) or GalNAc-T-myc (MDCK Clone 22) were dou- ble immunostained with antibodies to HA (green) and GM130, a Golgi apparatus mar- ker (red, top panels), or antibodies to myc (green) and GM130 (red, bottom panels). The right-hand panels show merged images from the glycosyltransferases (green) and GM130 (red). Single confocal sections were taken every 0.7 lm parallel to the coverslip. Scale bars ¼ 10 lm. (B) A schematic repre- sentation of the pathway of glycolipid bio- synthesis is shown on the left. Wild-type MDCK cells (MDCK wt) and cells from clones 13 (MDCK Clone 13) and 22 (MDCK Clone 22) were metabolically labelled with D-[U- 14 C]galactose for 24 h. Lipid extracts were purified, chromatographed on an HPTLC plate and visualized as indicated in Experimental procedures. The positions of co-chromatographed radioactive glycolipid standards are indicated (St). GM1 was also co-chromatographed and visualized by exposing the plate to iodine vapour. The position of GM1 is indicated on the right of the plate. Lipids migrated as multiple bands on the HPTLC plate because of the hetero- geneity of the fatty acyl chains of the mole- cules. (C) Wild-type MDCK cells (MDCK wt) and cells from clones 13 (MDCK Clone 13) and 22 (MDCK Clone 22) were labelled for GD3 with R24 antibody (green) or for GM1 with Alexa 555 -CTx b subunit (red). The image contrast in wild-type MDCK cells was reduced to show the presence of cells. Single confocal sections were taken every 0.7 lm parallel to the coverslip. Scale bars ¼ 10 lm. P. M. Crespo et al. Ganglioside trafficking in MDCK cells FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS 6045 We next attempted to characterize the expression and subcellular localization of GD3 and GM1, representa- tive gangliosides from the ‘a’ and ‘b’ series, for whose identification we have valuable and highly sensitive research methods. As shown in Fig. 1C, GD3 immunostaining with the mouse monoclonal antibody R24 (IgG3) [30] was typical of a plasma membrane con- stituent with a patchy distribution, as also observed in other cell lines [10]. A minor fraction was also observed in internal membranes. The expression of GD3 was below the limit of detection in wild-type MDCK cells, which only express GM3, thus confirming the specificity of the antibody. The expression of GM1 was evaluated by staining with the Alexa 555 -CTx b subunit, a protein that binds specifically and with a high affinity (k d =5· 10 )12 m) to the monosialoganglioside [31,32]. Interestingly, using this approach, we were able to detect the expression of GM1 in GalNAc-T-expressing MDCK cells (Fig. 1C). Thus, like GD3 expression, GM1 was predominantly expressed at the plasma membrane and no expression of GM1 was observed in wild-type MDCK cells. It should be mentioned that no apprecia- ble morphological alterations were observed in MDCK cells ectopically expressing either Sial-T2 or GalNAc-T. A B C Fig. 2. GM1 and GD3 gangliosides are apically localized in polarized MDCK cells. (A) The steady-state distribution of GM1 or GD3 in polar- ized MDCK cells was determined using immunofluorescence confocal microscopy. Polarized MDCK cells stably expressing GalNAc-T-myc (left panels) were fixed, permeabilized and incubated with CTx to label GM1 (CTx, green) and PI to label cell nuclei (PI, red), or with CTx to label GM1 (CTx, green) and fluorescent DiI to label plasma membrane (DiI, red). Polarized MDCK cells stably expressing Sial-T2-HA (right panels) were fixed, permeabilized and incubated with R24 antibody to label GD3 (R24, green) and PI to label cell nuclei (PI, red) or with R24 (R24, green) and DiI (DiI, red) to label plasma membrane. Cells were treated using laser scanning confocal microscopy with serial confocal sections (xy, 0.2 lm) collected from the top to the bottom of the cell monolayer. Then, xz sections were displayed using the ortho mode in LSM5 PASCAL software. The bottom rows in each set of images show merged images from CTx and R24 (green) with PI or DiI (red). The top (T) and bottom (B) of the cell monolayer are indicated. Scale bars ¼ 10 lm. (B) Domain-selective surface labelling of ganglioside in MDCK cells. GM1-expressing (left) and GD3-expressing (right) MDCK cells were grown on transmembrane filters to acquire polarity. Cells were then chilled and CTx or R24 was added to either the apical or basolateral chamber at 4 °C for 60 min. Next, cells from each filter were pro- cessed as indicated in Experimental procedures, and the amounts of CTx and R24 antibody bound to the apical or basolateral surface were analysed by western blotting. The expression of tubulin in the same membrane was analysed as a control of protein loading (Tub). (C) The relative contribution of bands in each condition was calculated using the computer software SCION IMAGE on the scanned film shown in (B). Ganglioside trafficking in MDCK cells P. M. Crespo et al. 6046 FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS GD3 and GM1 gangliosides are predominantly expressed at the apical surface of polarized MDCK cells Gangliosides are synthesized in the lumen of the Golgi apparatus by a complex system of membrane-bound glycolipid glycosyltransferases. After synthesis, ganglio- sides leave the Golgi apparatus via the lumenal surface of transport vesicles, which are mainly targeted to the plasma membrane [9]. More specifically, in polarized epithelial cells, gangliosides can be transported selec- tively to apical or basolateral surfaces, or can be homogeneously distributed in both specialized plasma membrane domains. To investigate the steady-state dis- tribution of GD3 and GM1 gangliosides in a polarized monolayer of MDCK clones, cells were grown to con- fluence and the expression of glycolipids was analysed by immunocytochemistry and fluorescent confocal microscopy. MDCK cells ectopically expressing either Sial-T2 or GalNAc-T manifested no noticeable morphological disruption of their epithelial organiza- tion and no altered transepithelial electrical resistance when grown on filter cultures (data not shown). Inter- estingly, both the disialoganglioside GD3 and the monosialoganglioside GM1 were mainly localized to the apical domain of MDCK cells (Fig. 2A, see top of the cell monolayer). To further confirm and quantify the steady-state distribution of GD3 and GM1 gangliosides in apical and basolateral domains of MDCK cells, we used a domain-selective labelling assay in which polarized MDCK cells grown on filter cultures were selectively labelled from either surface with the antibody R24 and CTx, specific for GD3 and GM1, respectively. The binding of R24 antibody and CTx in both apical and basolateral domains was evalu- ated by western blotting. In agreement with immuno- fluorescence labelling, we found that more than 90% of GM1 was selectively expressed at the apical surface (Fig. 2B,C). Approximately 70% of GD3 ganglioside was found to be expressed apically (Fig. 2B,C), compa- rable with the percentage observed for a glycosylphos- phatidylinositol (GPI)-anchored protein used as apical marker in MDCK cells [16]. Together, these results indicate that, after synthesis at the Golgi complex, both GD3 and GM1 gangliosides are preferentially transported to the apical surface of polarized MDCK cells. GD3 and GM1 gangliosides are rapidly endocytosed in MDCK cells It has been observed in different cell types that gangliosides can undergo endocytosis after arrival at the plasma membrane [8,33]. It has also been observed in MDCK cells that apically delivered cargos, such as the raft-associated HA of influenza virus, are very poorly internalized [34]. To specifically explore whether plasma membrane-expressed GD3 and GM1 ganglio- sides undergo endocytosis in MDCK cells, we used an antibody- and CTx b subunit-binding technique to track the fate of GD3 and GM1, respectively, after their internalization. Briefly, subconfluent GD3- and GM1-expressing MDCK cells were incubated on ice for 10 min to inhibit intracellular transport, and then with R24 or the Alexa 555 -CTx b subunit on ice for 45 min. Afterwards, cells were washed extensively with cold buffer in order to remove unbound antibody and toxin, and the temperature was changed to 37 °Cin order to restore transport and thereby allow the endo- cytosis of GD3 and GM1 for different times. Confocal microscopy analysis revealed that shortly (15 min) after allowing endocytosis by shifting the temperature to 37 °C, GD3 and GM1 were found in vesicles all around the cytoplasm (Fig. 3A). Almost the same sub- cellular distribution for GD3 was observed at 30 and 45 min after shifting the temperature to 37 °C, associ- ated with a noticeable decrease in the fluorescence intensity at later times. In contrast, the GM1-CTx b subunit began to acquire a perinuclear distribution at 15 min. After 30 min at 37 °C, the intracellular pool of GM1 became more concentrated in the perinuclear region and the plasma membrane mark had almost disappeared (Fig. 3A). By double immunofluorescence, we demonstrated that the perinuclear region stained with the GM1-CTx b subunit colocalized with the area of staining of GM130 and GalNAc-T-YFP, markers of the Golgi apparatus [27,35] (Fig. 3B). However, no colocalization was observed between endocytosed GD3 and GalNAc-T-YFP at any given time (Fig. 3B). Endocytosis of R24 and the Alexa 555 -CTx b subunit seems to be specifically mediated by GD3 and GM1 gangliosides, as wild-type MDCK cells, which only express GM3 ganglioside, did not bind and internalize the two ligands (results not shown). In addition, endo- cytosis of apically expressed GD3 was observed in fully polarized MDCK cells (Fig. S1). To further characterize the intracellular structures decorated by GD3 and GM1 gangliosides, we performed colocalization experiments with markers of apical and basolateral early endosome (GTPase Rab5- GFP; GFP, green fluorescent protein), apical recycling endosome (GTPase Rab11a-GFP) and common recy- cling endosome [labelled with endocytosed Alexa 647 - transferrin (Tf)]. As shown in Fig. 3C, internalized GD3 is detected in apical early endosome, partially detected in apical recycling endosome, but not detected P. M. Crespo et al. Ganglioside trafficking in MDCK cells FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS 6047 GD3 A B C 0 min 15 min 30 min 45 min GM1 Fig. 3. GD3 and GM1-CTx are rapidly and specifically endocytosed in MDCK cells. (A) MDCK cells stably expressing GD3 (GD3, top panels) or GM1 (GM1, bottom panels) were incubated with R24 or Alexa 555 -CTx b subunit, respectively, at 4 °C. The temperature was then shifted to 37 °C to allow endocytosis of GD3-R24 and GM1-CTx, and cells were fixed at 0, 15, 30 or 45 min and processed for immunostaining. Sin- gle confocal sections (xy) of 0.7 lm were taken parallel to the coverslip. Scale bars ¼ 10 lm. (B) Left panel: MDCK cells stably expressing GD3 and transiently expressing GalNAc-T-YFP, a Golgi apparatus marker (pseudo-coloured green), were allowed to internalize R24 antibody for 30 min. Then, R24 antibody was visualized using Alexa 546 -conjugated goat anti-mouse IgG (red). Middle panel: MDCK cells stably expressing GM1 and transiently expressing GalNAc-T-YFP (pseudo-coloured green) were allowed to internalize Alexa 555 -CTx b subunit for 30 min (red). Right panel: cells were processed as shown in the middle panel, except that they were immunostained with antibody to GM130, another Golgi apparatus marker (green). (C) MDCK cells stably expressing GD3 (top panels) or GM1 (bottom panels) were allowed to internalize R24 antibody (red) or Alexa 555 -CTx b subunit (red), respectively, for 15 min (left panels) or 45 min (middle and right panels). As indicated, the cells were transiently transfected to express wild-type Rab5-GFP (left panels, green) or wild-type Rab11a-GFP (middle panels, green) or allowed to internalize Alexa 647 -Tf (Tf-Alexa 647 , pseudo-coloured green). Insets in the merged panels show the details at higher mag- nification. Single confocal sections (xy) of 0.7 lm were taken parallel to the coverslip. The expression of fusion proteins and endocytosed Tf-Alexa 647 was analysed by the intrinsic fluorescence. Scale bars ¼ 10 lm. Ganglioside trafficking in MDCK cells P. M. Crespo et al. 6048 FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS in common recycling endosome at any given time. Like GD3, GM1 was also observed to colocalize with wild-type Rab5 and Rab11a (Fig. 3C), as well as with common recycling endosome (Fig. 3C) and the Golgi apparatus (Fig. 3B). It is probable that, in analogy with the results obtained in other cells [31,32,36], inter- nalized CTx can traffic to apical early endosome from where it is differentially sorted. A fraction is recycled back to the plasma membrane via the apical recycling endosome (Rab11a positive); the remainder is sorted to the Golgi apparatus probably via the common recycling endosome. The association of internalized GD3-R24 with the apical early endosome and apical recycling endosome in MDCK cells is compatible with its probable recy- cling back to the plasma membrane, as recently observed in nonpolarized epithelial CHO-K1 cells [33]. To explore this hypothesis, GD3-expressing MDCK cells were incubated on ice for 10 min to inhibit intra- cellular transport, and then with R24 antibody at 4 °C for 60 min. Afterwards, cells were allowed to interna- lize the antibody for 30 min at 37 °C, and then the temperature was shifted again to 4 °C. The cell surface was then stripped of any remaining antibody with an acid wash (0 min). Finally, the cells only contained R24 antibody in intracellular compartments. Subse- quently, prewarmed culture medium was added to the cells and they were maintained at 37 °C to restore intracellular transport. Cells and culture medium were recovered at different times, and the presence of the R24 antibody in both samples was analysed by western blotting. As shown in Fig. 4, at the beginning of the time-course experiment (stripped cells, 0 min), the anti- body was present only in the cell fraction. At 15 min, the antibody was detected in both fractions (cells and culture medium), and, at 60 min, most of the R24 anti- body was recovered from the culture medium. The antibody recovered from the culture medium was found to have the expected molecular mass (whole molecule) in gels run under nonreducing conditions. Together, these results indicate that GD3-R24 anti- body, once internalized in MDCK cells, is recycled back to the plasma membrane and released to the culture medium. Clathrin is not required for the internalization of GD3 and GM1 gangliosides in MDCK cells Previously, we have described, in nonpolarized epithe- lial CHO-K1 cells, that endocytic recycling of GD3 is sensitive to Brefeldin A [33], consistent with the requirement of clathrin-coated vesicles for efficient GD3 recycling. To further explore the early endocytic process involved in the internalization of GD3 in A B Fig. 4. R24 antibody is recycled back to the plasma membrane and released to the culture medium. (A) GD3-expressing MDCK cells were incubated with R24 antibody for 60 min on ice. Afterwards, cells were allowed to internalize the antibody for 30 min at 37 °C, and the tem- perature was then shifted again to 4 °C. The cell surface was then stripped of any remaining antibody with an acid wash (0 min). The cells were then incubated at 37 °C to restore intracellular transport, and cells and the culture medium were recovered at 15, 30 and 60 min. The presence of R24 antibody in both samples was analysed by western blotting under nonreducing conditions, as indicated in Experimental pro- cedures. (B) The relative contribution of the bands in each condition was calculated using the computer software SCION IMAGE on the scanned film shown in (A). Tubulin expression was used to normalize the level of proteins seeded in each lane. The band intensity for R24 antibody at 0 min (cellular fraction) was arbitrarily taken as unity. The results are representative of three independent experiments. P. M. Crespo et al. Ganglioside trafficking in MDCK cells FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS 6049 MDCK cells, we used a dominant-negative mutant of epidermal growth factor receptor pathway substrate clone 15 (Eps15), which selectively affects clathrin- mediated endocytosis. Eps15 is an established compo- nent of clathrin-coated pits that is ubiquitously and constitutively associated with adaptor proteins; the expression of dominant-negative Eps15 selectively inhibits clathrin-mediated endocytosis [37]. First, we demonstrated that the expression of dominant-negative Eps15 in subconfluent GD3-expressing MDCK cells inhibits the internalization of Alexa 647 -Tf, which is known to be a clathrin-dependent process [13,38] (Fig. 5A). Next, subconfluent GD3-expressing MDCK cells transiently expressing dominant-negative Eps15 were incubated on ice with the R24 antibody for 45 min. The cells were then washed to remove unbound antibody, prewarmed culture medium was added and the cells were transferred to 37 °C to allow endocytosis for 30 min. The results shown in Fig. 5B indicate that the expression of dominant-negative Eps15 did not affect GD3 internalization, as the frac- tions of internalized and accumulated antibody at 30 min were similar in both control and dominant- negative Eps15-expressing cells. This results indicate that clathrin-coated vesicles do not participate in the endocytic process of GD3 in MDCK cells. A B C Fig. 5. GD3 and GM1-CTx are internalized in MDCK cells by clathrin-independent endocytosis. (A) GD3-expressing MDCK cells were tran- siently transfected to express the dominant-negative form of Eps15-GFP (DN-Eps15-GFP, green). After 24 h, uptake of Alexa 647 -Tf (Tf-Alexa 647 , pseudo-coloured red) was monitored for 20 min. (B) GD3-expressing MDCK cells were transiently transfected to express the dominant-negative form of Eps15-GFP (DN-Eps15-GFP, green). After 24 h, the cells were incubated with R24 antibody to GD3 at 4 °C, and then allowed to internalize the antibody for 30 min by shifting the temperature to 37 °C. Cells were fixed and the presence of R24 was anal- ysed using Alexa 546 -conjugated goat anti-mouse IgG (GD3, red). (C) GM1-expressing MDCK cells were transiently transfected to express the dominant-negative form of Eps15-GFP (DN-Eps15-GFP, green). After 24 h, cells were incubated with the Alexa 555 -CTx b subunit at 4 °C, and then allowed to internalize the complex GM1-TCx for 15 min by shifting the temperature to 37 °C. Cells were fixed and the presence of CTx was analysed by confocal microscopy (GM1, red). Single confocal sections (xy) of 0.7 lm were taken parallel to the coverslip. Arrows indicate DN-Eps15-GFP-transfected cells. The expression of DN-Eps15-GFP and endocytosed Tf-Alexa 647 was analysed by the intrinsic fluorescence. Right panels show merged images from the two acquired channels. Scale bars ¼ 10 lm. Ganglioside trafficking in MDCK cells P. M. Crespo et al. 6050 FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS It has been demonstrated that GM1-CTx can exploit three different internalization pathways en route to the Golgi apparatus and endoplasmic reticulum [39]. GM1- CTx can be internalized by a nonclathrin-mediated, noncaveolar, cholesterol-sensitive pathway or by clath- rin-mediated endocytosis or by caveolae. Following essentially the experimental protocol described above for the internalization of GD3, we observed that the GM1-Alexa 555 -CTx b subunit is internalized in domi- nant-negative Eps15-expressing MDCK cells (Fig. 5C). Thus, these results suggest that GM1-CTx internaliza- tion in MDCK cells occurs through a clathrin-indepen- dent process, or that clathrin-mediated internalization is a relatively minor pathway in these epithelial cells. Discussion We have developed a panel of cell lines derived from MDCK cells whose glycolipid composition has been genetically modified by the transfection of key ganglio- side glycosyltransferases. MDCK cells express GM3 as the sole ganglioside species at the cell surface. Trans- fection with GalNAc-T or Sial-T2, two enzymes that act on GM3 at the branching point of the synthesis pathway, directs the flow of GM3 to either GM2 and series ‘a’ gangliosides [40] or to GD3 (series ‘b’) and GT3 (series ‘c’) [9], respectively. In addition, the expression of GalNAc-T resulted in the synthesis of GA2, a neutral glycolipid belonging to series ‘o’, by catalysing the transfer of N-acetyl-d-galactosamine to the galactose residue of LacCer. As observed previ- ously in other cell lines [9,41], it is expected that the synthesis in the Golgi apparatus, intracellular transport and topological disposition of the new ganglioside species in the plasma membrane in transfected MDCK cells will follow the physiological mechanisms estab- lished in the parental cells. In this report, we have focused on the examination of the subcellular distribution and intracellular trans- port of GD3 and GM1, representative complex gangliosides from series ‘a’ and ‘b’, respectively. Using biochemical and fluorescent confocal microscopy, we have demonstrated that GD3 and GM1 gangliosides, after synthesis at the Golgi apparatus, are mainly delivered to the apical plasma membrane of polarized MDCK cells. It is known that, for proteins, apical sorting signals are localized in exoplasmic, membrane or cytoplasmic domains, and comprise moieties as divergent as lipids (GPI), sugars (N- and O-glycans) and peptide motifs located at both the transmembrane and cytoplasmic domains [13]. Much evidence has demonstrated that some of these apical sorting motifs mediate the incorporation of proteins into lipid rafts, membrane microdomains that are enriched in choles- terol and sphingolipids, a hypothesis put forward by van Meer and Simons [19]. According to the lipid raft hypothesis, lipids rafts and their associated proteins form sorting platforms at the TGN that are incorpo- rated into apical transport intermediates and delivered to the apical surface. More recently, it has been reported that protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins [42]. Do GD3 and GM1 gangliosides associate with lipid rafts for trafficking to the apical surface? It is accepted that some biochemical properties associated with polar- ized pathways are preserved in fibroblast cell lines [43]. In this sense, we found that newly synthesized ganglio- sides, including GD3 and GM1, did not partition into lipid raft domains in the Golgi apparatus of nonpolar- ized CHO-K1 cells [9,10]. Nevertheless, the presence of GD3 in lipid rafts was also evaluated in MDCK cells according to the usual criterion of insolubility in non- ionic detergent at 4 °C. We observed that membrane- expressed GD3 was about 70% soluble to detergent extraction (Fig. S2). Comparatively, GM3, an endoge- nously synthesized ganglioside enriched in the basolat- eral domain of MDCK cells [44], partitioned in equal percentages between the Triton-X100-soluble and Tri- ton-X100-insoluble fractions in both wild-type and GD3-expressing MDCK cells. Thus, the partitioning of GD3, and probably GM1, into lipid rafts does not account for the polarized distribution of this glycolipid in MDCK cells. Further work using biophysical tech- niques, which measure lipid raft association more accu- rately than does detergent insolubility, is required. However, progress in this area is slow because of tech- nical difficulties with available fluorescent lipids. Short- chain and fluorescent lipid analogues have been employed as useful tools to delineate the potential pathways and mechanisms of intracellular transport and sorting [45]. Nevertheless, quantitative comparison with natural lipids remains to be determined, as it has been reported previously that the quantitative and qualitative behaviour of analogous lipids is quite differ- ent from that of long-chain cellular lipids [46]. In the current study, we have shown that GD3 ganglioside, in association with a specific antibody, is actively endocytosed in subconfluent and polarized MDCK cells. Endocytosed GD3 was detected in both Rab5-positive apical early endosome and Rab11a-posi- tive apical recycling endosome, which is compatible with its recycling back to the plasma membrane, as recently observed in nonpolarized CHO-K1 cells [33]. Indeed, this hypothesis was confirmed in MDCK cells. GD3-R24 antibody was rapidly endocytosed, recycled P. M. Crespo et al. Ganglioside trafficking in MDCK cells FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS 6051 back to the plasma membrane and released to the cul- ture medium. At this point, we cannot discard the recycling of the GD3-R24 complex to the plasma membrane from Rab5-positive apical early endosome through the direct return pathway. Interestingly, the endocytosis of GD3 in MDCK cells was found to be a clathrin-independent process, in line with previous results showing the caveolae-mediated endocytosis of glycosphingolipid analogues [47]. However, the mecha- nism(s) of apical endocytosis of GD3 ganglioside in MDCK cells remains to be investigated, as caveolae are formed only at the basolateral surface [13]. It should be mentioned that sorting and recycling endo- some compartments have an acidic pH (6–6.5), which could dissociate the ganglioside–antibody complex. However, we demonstrated that the association of antibody R24 with GD3 ganglioside was only slightly affected, even after 1 h at pH 6 or 7 (Fig. S3), suggest- ing that the itinerary of the R24 antibody reflects the intracellular transit of the complex. It has been demonstrated in different cell types that CTx can be internalized by clathrin-, caveolar- and CDC42-dependent pathways en route to the endoplas- mic reticulum [47,48]. In the present study, we observed that GM1-CTx can be efficiently endocytosed in dominant-negative Eps15-expressing MDCK cells, suggesting that GM1-CTx internalization occurs through a clathrin-independent process, or that clath- rin-mediated internalization is a relatively minor path- way in these epithelial cells. After GM1-CTx internalization, we essentially observed that it follows the intracellular itinerary described for other cell types (plasma membrane fi endosomes fi Golgi appara- tus). However, in MDCK cells, we observed that the binding of CTx mainly occurs in the apical domains, whereas, in polarized CaCo-2 cells, the endocytosis of CTx is essentially the same at both apical and basolat- eral surfaces [49]. As also observed for the GD3–anti- body complex, the association of CTx with the monosialoganglioside GM1 was not affected at pH 6 (Fig. S3), suggesting that the intracellular trafficking of CTx through acidic compartments occurs in associa- tion with GM1. Moreover, it has been suggested that the monosialoganglioside is the vehicle that directs the toxin to its final destination [31,48]. In conclusion, our results demonstrate, for the first time, that complex gangliosides are apically sorted in polarized MDCK cells, and that GD3 and GM1 gan- gliosides are internalized by clathrin-independent endo- cytosis to follow different intracellular destinations. A potential function of ganglioside recycling involves the selective endocytosis of particular cell surface compo- nents during the generation and maintenance of mem- brane polarity. A further potential role involves the regulation of signal transduction processes. Previous work has reported that cell surface receptors and sig- nalling molecules are recruited into specialized mem- brane domains enriched in glycosphingolipids, and that gangliosides modify the biological effects of sev- eral trophic factors [1,4,50]. Consequently, the internal- ization of these domains may well play an important role in signalling events. GD3 and GM1 internalization and trafficking in MDCK cell lines were monitored using a specific anti- body to GD3 and CTx, respectively. Therefore, the results described in this work provide the basis to gain further insight into the molecular mechanisms that operate in the intracellular trafficking and pathological effects of bacterial toxins and antibodies to ganglio- sides, which are associated with autoimmune disorders [51,52]. In addition, the genetically modified MDCK cell lines expressing both complex gangliosides and neutral sphingolipids should provide excellent model systems in which to study the synthesis, trafficking and polarity of glycolipids in epithelial cells. Experimental procedures Expression plasmids The expression plasmid for SialT-2-HA (carboxy-terminal epitope-tagged chicken SialT-2 with the nanopeptide epi- tope of viral HA; pCEFL-SialT-2-HA) has been described previously by Daniotti et al. [53]. GalNAc-T-myc (3¢ end of the human GalNAc-T cDNA epitope tagged with human c-myc; pCIneo-GalNAc-T-myc) has been described previ- ously by Giraudo et al. [54]. The GTPase Rab11a-GFP wild-type construct was kindly provided by M. Colombo (Universidad Nacional de Cuyo, Mendoza, Argentina); the plasmid coding for Rab5-GFP was supplied by J. Bonifa- cino (NICHD, National Institutes of Health, Bethesda, MD, USA). The construct containing the cDNA coding for the N-terminal domain (cytosolic tail, transmembrane domain and few amino acids of the stem region) of Gal- NAc-T fused to the N-terminus of YFP (GalNAc-T-YFP) was obtained by subcloning the corresponding cDNA frag- ments into the plasmid pEYFP-N1 (Clontech, Mountain View, CA, USA) [55]. Cell culture and generation of stable MDCK cell lines MDCK II cells (generously provided by A. Gonza ´ lez, Pontificia Universidad Cato ´ lica de Chile, Chile) were main- tained in DMEM (Gibco BRL, Carlsbad, CA, USA) sup- plemented with 7.5% fetal bovine serum (HyClone, Logan, Ganglioside trafficking in MDCK cells P. M. Crespo et al. 6052 FEBS Journal 275 (2008) 6043–6056 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... Paladino S, Pocard T, Catino MA & Zurzolo C (2006) GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells J Cell Biol 172, 1023–1034 16 Keller P, Toomre D, Diaz E, White J & Simons K (2001) Multicolour imaging of post-Golgi sorting and trafficking in live cells Nat Cell Biol 3, 140–149 17 Ait Slimane T & Hoekstra D (2002) Sphingolipid trafficking and protein sorting... extractability of in uenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts J Cell Biol 108, 821–832 Nichols GE, Shiraishi T & Young WW Jr (1988) Polarity of neutral glycolipids, gangliosides, and sulfated lipids in MDCK epithelial cells J Lipid Res 29, 1205–1213 Pagano RE (2003) Endocytic trafficking of glycosphingolipids in sphingolipid storage... stable MDCK II cells expressing SialT-2 or GalNAc-T, cells were transfected with pCEFL-SialT-2-HA or pCIneo-GalNAc-T-myc using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA, USA) Following 48 h of expression, cells were selected with 0.8 mgÆmL)1 G418 (Invitrogen) Resistant colonies were picked up with cloning cylinders, expanded and screened for GD3 or GM1 expression All cell lines were maintained under... receptor class B type I in MDCK cells Proc Natl Acad Sci USA 101, 3845–3850 Supporting information The following supplementary material is available: Fig S1 GD3 ganglioside is endocytosed in polarized MDCK cells Fig S2 Metabolic labelling and Triton-X100 extraction of total glycolipids expressed in wild-type and genetically modified MDCK cells Fig S3 Effect of pH on GD3-R24 antibody and GM1-CTx association... cells were incubated on ice for 10 min to inhibit intracellular transport, and then with R24 antibody for 60 min on ice The cells were then transferred to 37 °C for 30 min to allow R24 endocytosis Cell surfacebound antibody was then removed by acid stripping at 4 °C and the cells were washed extensively with cold NaCl ⁄ Pi The cells were then incubated at 37 °C with 1 mL of prewarmed fresh DMEM in order... Afterwards, cells were washed three times with cold NaCl ⁄ Pi, transferred to 37 °C with fresh prewarmed complete DMEM to allow antibody and CTx internalization for different times, and finally fixed in 4% paraformaldehyde in NaCl ⁄ Pi at room temperature for 30 min For Tf internalization, cells were first incubated for 90 min in DMEM without fetal bovine serum, next incubated at 4 °C in cold DMEM containing... containing 20 mm Tris ⁄ HCl, 150 mm NaCl, 1 mm EDTA, pH 7.5, 1% Triton-X100 and protease inhibitor cocktail Proteins from the lysates were precipitated with chloroform–methanol (1 : 4, v ⁄ v) and resuspended in NaCl ⁄ Pi R24 antibody and CTx bound to apical or basolateral surface domains were assessed by western blotting Metabolic labelling, lipid extraction and chromatography GD3-expresing MDCK cells. .. sorting in epithelial cells FEBS Lett 529, 54–59 18 Gut A, Balda MS & Matter K (1998) The cytoplasmic domains of a beta1 integrin mediate polarization in Madin–Darby canine kidney cells by selective basolateral stabilization J Biol Chem 273, 29381–29388 19 van Meer G & Simons K (1988) Lipid polarity and sorting in epithelial cells J Cell Biochem 36, 51–58 20 Simons K & Ikonen E (1997) Functional rafts in. .. receptors by gangliosides Sci STKE 2002, RE15 5 Proia RL (2003) Glycosphingolipid functions: insights from engineered mouse models Philos Trans R Soc London B: Biol Sci 358, 879–883 6 Hakomori S, Handa K, Iwabuchi K, Yamamura S & Prinetti A (1998) New insights in glycosphingolipid function: ‘glycosignaling domain’, a cell surface assembly of glycosphingolipids with signal transducer molecules, involved in. .. levels of protein expression [56] Cell labelling and internalization assays MDCK II cells transiently transfected or not with the plasmids indicated above were incubated on ice for 20 min to inhibit intracellular transport Then, the cells were incubated on ice for 60 min with hybridoma [American Type Culture Collection (ATCC) No HB-8445] supernatant containing R24 antibody or 6 lgÆmL)1 Alexa555-CTx b . Complex gangliosides are apically sorted in polarized MDCK cells and internalized by clathrin-independent endocytosis Pilar M. Crespo,. apparatus. In conclusion, our results demonstrate that complex gangliosides are apically sorted in polarized MDCK cells, and that GD3 and GM1 gangliosides are internalized