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Vol. 18, Issue 6, 2057-2071, June 2007
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*Department of Cell and Animal Biology and Confocal Unit, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel; Experimental Medicine, Universite Catholique de Louvain and Christian de Duve Institute of Cell Pathology, B-1200 Brussels, Belgium; and School of Engineering, Bar Ilan University, Ramat Gan 52900, Israel
Submitted August 22, 2006;
Revised March 15, 2007;
Accepted March 21, 2007
Monitoring Editor: Keith Mostov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Rojas and Apodaca, 2002) and is carried from the blood to mucosal secretions by the polymeric immunoglobulin receptor (pIgR). The itinerary of pIgR and its ligand was intensively studied in the polarized Madin-Darby canine kidney (MDCK) cell line that heterologously expresses rabbit pIgR (for a recent review, see Rojas and Apodaca, 2002). The receptor is initially delivered from the trans-Golgi network (TGN) to the basolateral plasma membrane, where it encounters and binds dIgA. The complex is subsequently internalized and transcytosed to the apical plasma membrane of the cell. At that surface, the ectodomain of pIgR is proteolytically cleaved off to yield the secretory component (SC), which is released together with dIgA into mucosal secretions. In the course of transcytosis, pIgR-dIgA complexes traverse various endosomal compartments, among which is an endocytic compartment located immediately underneath the apical surface (Gibson et al., 1998). This apical compartment, defined as apical recycling endosomes (AREs), is enriched in dIgA and is relatively deficient in recycling transferrin (Apodaca et al., 1994; Barroso and Sztul, 1994; Brown et al., 2000). AREs are thought to play a unique role in transcytosis, possibly by exploiting apical recycling mechanisms to shuttle transcytotic cargo to the apical surface.
DIgA binding to pIgR on the basolateral surface stimulates transcytosis of rabbit pIgR expressed in MDCK cells (Song et al., 1994) and in rat liver in vivo (Giffroy et al., 1998). Ligand-mediated receptor dimerization (Singer and Mostov, 1998) might be required to elicit the subsequent intracellular signaling events contributing to the regulation of transcytosis. Within 10 s after dIgA binding, several cellular proteins (but not pIgR itself) become tyrosine-phosphorylated (Luton et al., 1998). This early and transient event could be mediated by the recruitment of the nonreceptor tyrosine kinase p62yes (Luton et al., 1999). The most important findings suggested that stimulation of transcytosis by dIgA requires pIgR sensitization by ligand binding at the basolateral surface and signal transduction to the apical cytoplasm (Luton and Mostov, 1999). Collectively, these observations argued that signaling events originating from the basolateral surface and/or basolateral early endosomes are essential for the stimulation of dIgA transcytosis.
A growing body of evidence indicates that a significant portion of the signal transduction pathways takes place via plasma membrane receptors positioned at specialized microdomains, termed lipid rafts, which are enriched in cholesterol and glycosphingolipids (Moffett et al., 2000; Simons and Toomre, 2000; Waugh et al., 2001; Inoue et al., 2002). Here we hypothesized that dIgA binding stimulates the insertion of pIgR into cholesterol-rich domains (lipid rafts) located on the basolateral surface and/or basolateral endosomes of polarized MDCK cells. This event could play a pivotal role in the sorting of dIgA-pIgR complexes into the transcytotic pathway. However, previous experiments that attempted to address this hypothesis failed to yield consistent results (Hansen et al., 1999; Sarnataro et al., 2000; De Marco et al., 2002). In this study, we directly investigated the functional significance of membrane cholesterol in polarized traffic after endocytosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cyclodextrin (mCD, average substitution: 10.5–14.7 methyl groups per molecule, C-4555), its inactive analog 
-cyclodextrin (CD, C-4642), and cholesterol (C-8503) were from Sigma (St. Louis, MO). Iodine-125 (125I) and [35S]Cys/Met (PRO-MIX) were from Amersham Biosciences (Amersham, United Kingdom).
Proteins and Antibodies
Purified human dimeric IgA (dIgA) antibodies were manufactured by J.-P. Vaerman. Human apo-transferrin (hTfn; Biological Industries, Beit Haemek, Israel) was loaded with iron as described (Podbilewicz and Mellman, 1990). Anti-human transferrin receptor (hTfnR) antibody (H68.4), sheep anti-rabbit SC antibodies, and monoclonal SC166 antibody directed against the pIgR's cytoplasmic tail have been described elsewhere (Solari et al., 1985; White et al., 1992; Aroeti and Mostov, 1994). Fluorescein isothiocyanate (FITC)-labeled iron-loaded hTfn and AlexaFluor 594 donkey anti-rabbit and AlexaFluor 488 goat anti-mouse antibodies were from Molecular Probes (Eugene, OR). Different antibodies have been used for detecting caveolin-1 (Cav1). Rabbit anti-Cav1 (610059, BD Transduction Laboratories, Lexington, KY) was used for immunofluorescence analyses. Mouse anti-Cav1 clone 2297 (BD Transduction Laboratories) and rabbit anti-Cav1 (C-3237, Sigma, St. Louis, MO) were used for detecting Cav1 in Western blotting. Mouse anti-Cav1 (pY14), phospho-specific (clone 56) from BD Transduction Laboratories was used for detecting Cav1 phosphorylated on tyrosine 14 (pY14-Cav1). Monoclonal anti-calnexin antibodies were from StressGen Biotechnologies (San Diego, CA). The rat anti-zonula occludens-1 (ZO1) R40.76 antibodies (Dr. D. A. Goodenough, Harvard University, Cambridge, MA) were used to label MDCK tight-junctions. FITC-conjugated affinity-purified goat antibodies to human IgA were from Sigma. AffiniPure rabbit anti-human serum IgA and -chain–specific antibodies were from Jackson ImmunoResearch Labs (West Grove, PA).
Cell Culture
MDCK and PTR-MDCK cells (MDCK coexpressing rabbit pIgR and hTfnR; Brown et al., 2000; Wang et al., 2000) were cultured routinely in 10-cm dishes, in minimal essential medium (MEM, Biological Industries) containing 5% fetal calf serum (Biological Industries) and 1% antibiotics (Biological Industries). To induce polarity, cells were cultured on polycarbonate filter supports (0.4-µm pore size, Transwell, Costar, Cambridge, MA) for 4 d before each experiment (Aroeti et al., 1993). For flotation experiments, fungicides were omitted from the growth medium for at least 24 h before the experiment. Cell monolayer integrity was estimated by monitoring the transepithelial electrical resistance, using a Millicel-ERS-system (Millipore, Bedford, MA), equipped with a silver/silver-chloride electrode. The value from a blank filter (with no cells, but otherwise treated identically) was subtracted from the resistance values of filters on which cells were plated.
Cholesterol Depletion and Enrichment
The relevance of cholesterol-rich microdomains in cellular processes is typically examined by altering cholesterol levels in cell membranes. The drug cyclodextrin specifically disrupts lipid rafts by chelating cholesterol from cells (Kilsdonk et al., 1995; Yancey et al., 1996). For acute cholesterol depletion of cellular membranes, filter-cultured PTR-MDCK cells were treated for different times, not longer than 60 min at 37°C with 10 mM mCD, dissolved in MEM containing bovine serum albumin (MEM/BSA: MEM containing HBSS, 20 mM HEPES, pH 7.4, and 0.6% BSA). The agent was applied simultaneously into the apical and basolateral chambers of the Transwell support. MCD saturated with cholesterol (mCD/chol) was prepared as previously described (Grimmer et al., 2000). Cholesterol enrichment of cellular membranes was achieved by treating the cells for 60 min at 37°C with 10 mM mCD/chol dissolved in MEM/Hanks' salts lacking BSA. Cellular cholesterol levels were determined for confluent cell cultures grown in 2 x 5-cm dishes. Cells were solubilized in SDS buffer (0.1% SDS, 1 mM EDTA, 0.1 M Tris·Cl, pH 7.4) at 37°C and cholesterol levels were determined using the Infinity cholesterol reagent kit (Sigma). Cholesterol content was normalized to the lysate's protein levels (determined by the microbicinchoninic acid reagent kit; Pierce, Rockford, IL). Typically, cell treatment with mCD at 37°C reduced the cellular cholesterol content by 30–40% of the control. Cell exposure to mCD/chol resulted in elevated cholesterol levels for up to 
60% of the control.
Flotation of Detergent-resistant Membranes
Detergent-resistant membranes (DRMs) were isolated as previously described (Brown and Rose, 1992) with some modifications (Naslavsky et al., 1997). Cells cultured on 75-mm polycarbonate filters were lysed in the cold with ice-cold buffer containing 1% Triton X-100 (10 mM Tris, pH 7.5, 10 mM EDTA, 100 mM NaCl, and 1% Triton X-100) supplemented with protease and phosphatase inhibitors. Lysates were homogenized by 10 passages through a 25-gauge needle and subsequently adjusted to 35% Nycodenz (D-2158, Sigma) dissolved in ice-cold TNE buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA). The solution was loaded at the bottom of a TLS-55 ultracentrifuge tube. An 8–35% Nycodenz linear step gradient was overlaid above the lysate-containing layer, and after centrifugation for 4 h at 55,000 rpm in a TLS-55 Beckman rotor (Fullerton, CA) at 4°C, 12 180-µl fractions were collected from the top. Proteins in each fraction were precipitated by methanol/chloroform and analyzed by SDS-PAGE and Western blotting. A third of the protein quantity in each of fractions 9–12 was analyzed. The partitioning of a given protein with DRMs and detergent-soluble fractions was based on comparing its flotation profile with the classical lipid raft protein Cav1 and the nonraft marker calnexin, respectively. Cav1 was typically distributed within fractions 1–6 [designated DRMs], whereas calnexin was distributed mainly in fractions 7–12, representing the detergent soluble lysate (see Figure 1A).
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Detection of hTfnRs in Basolateral DRMs
The basolateral surface of 75-mm filter-grown PTR-MDCK cells was biotinylated in the cold as described above. In some experiments, biotinylated surface proteins were allowed to internalize by incubating the cells at 37°C for various times. After quenching of residual biotin, cells were lysed in buffer containing Triton X-100. A flotation assay for isolation of DRMs was performed, and biotinylated proteins in each fraction were pulled down with streptavidin-agarose beads (S-1638, Sigma). After analysis by SDS-PAGE and Western blotting, hTfnR was detected by probing the nitrocellulose membranes with H68.4 monoclonal antibodies directed against its cytoplasmic domain.
Immunofluorescence Analysis
Cells were fixed with 4% paraformaldehyde in PBS, pH 8.0 (10 min at 4°C followed by 20 min at 22°C); the remaining active sites were quenched with 75 mM NH4Cl + 20 mM glycine in PBS, pH 8.0, and cells were subsequently processed for immunofluorescence and confocal microscopy, as previously described (Orzech et al., 1999). For immunolabeling of pY14-Cav1, cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS, pH 8.0, after fixation, and subsequent steps were performed as above.
Confocal images were acquired either with a Bio-Rad MRC1024 (Richmond, CA) coupled to a Zeiss Axiovert 135M (Thornwood, NY), or on an Olympus FV1000 (Melville, NY). With either system, two excitation lasers were used sequentially: 488 nm for the FITC or Alexa488 and either 543 nm (FV1000) or 594 nm (MRC1024) for the Alexa594. A narrow-band emission filter (505–525 nm for the FV1000, 505–545 nm for the MRC1024) was used in the FITC channel. Sequential frame acquisition was used to eliminate cross-talk between the two fluorescence channels. Colocalization was characterized using the method of Lachmanovich et al. (2003). Briefly, the peaks were segmented using a local threshold set at half the maximum value of the local peak. Objects were considered colocalized if the center of mass of one class fell within the area of the second. Results were compared with a binomial model, and randomized images were used to verify that random distributions of staining produced the expected value as predicted by the binomial distribution.
It should be stressed that the colocalization measurement is only meaningful to the extent that the result differs significantly from that which would be expected from a random distribution of the fluorescent objects.
Radio-iodination of Ligands
Iron-saturated hTfn and dIgA were radioiodinated to a specific activity of 5–9 x 106 cpm/µg using the iodine monochloride method (Breitfeld et al., 1989).
Trafficking Assays
Analysis of Endocytosis.
Endocytosis of radioiodinated ligands was measured as previously described (Okamoto et al., 1992). A thiol-cleavable biotin compound (sulfo-NHS-SS-biotin, Pierce) was used as an alternative approach to determine internalization of unoccupied receptors (Okamoto et al., 1994).
Analysis of 125I-hTfn Recycling and Transcytosis.
The fate of a single cohort of basolaterally internalized 125I-hTfn was monitored principally as described (Gan et al., 2002). Briefly, 125I-hTfn was internalized from the basolateral side of 12-mm filters at 37°C. After several washes at 17°C with MEM/BSA, cells were treated for 30 min at 37°C with cyclodextrin. In some experiments, cholesterol depletion preceded the ligand internalization step. Surface-bound ligand was stripped off with a buffer containing 50 mM MES, pH 5.0, and 200 mM NaCl for 2 min. Cells were then incubated at 37°C with plain MEM/BSA containing 3 µg/ml unlabeled hTfn and 100-µM deferoxamine mesylate (an iron chelator, D-9533, Sigma). Radioactivity was measured using a 
-counter, as described (Gan et al., 2002).
Analysis of 125I-dIgA Recycling and Transcytosis.
Transcytosis of 125I-dIgA was monitored as previously described (Brown et al., 2000), with some modifications. To ensure a follow-up of 125I-dIgA traffic from endosomes, the ligand was internalized at 37°C. Unbound ligand was removed by washing at 17°C. Cells were then exposed to mCD for 30 min at 37°C. Surface-associated ligand was removed by treating the cells with the MES, pH 5.0/NaCl buffer for 60 min at 4°C, and the ligand was chased in MEM/BSA at 37°C. In some cases, cell treatment with mCD preceded the ligand internalization step. Radioactivity was measured using a -counter, as described (Aroeti and Mostov, 1994).
Analysis of Biotinylated pIgR Transcytosis.
Constitutive transcytosis of the empty pIgR was monitored as described (Aroeti and Mostov, 1994). Cells grown on 24-mm filters were metabolically labeled with [35S]Cys/Met for 45 min at 37°C. Proteins at the basolateral surface were biotinylated at 37°C (0.1-mg/ml sulfo-NHS-LC biotin). In some experiments, cyclodextrin treatment (incubation for 30 min at 37°C in MEM/BSA containing mCD) preceded the cell surface biotinylation step, whereas in other experiments cyclodextrin treatment was applied after biotinylation. Receptor traffic was promoted by further incubating the cells for 30 or 60 min at 37°C in MEM/BSA. Biotinylated pIgR and SC were immunoprecipitated (IPed) from filters and media, respectively. The IPed material was eluted by resuspending the immunobeads in 10% SDS and heating for 3 min at 95°C. Biotinylated pIgR and SC were recovered from the eluate using streptavidin-coated agarose beads. Radioactive proteins were analyzed by SDS-PAGE. Protein bands were visualized and quantified by the Fuji FLA 3000 apparatus (Tokyo, Japan).
Analysis of Biotinylated TfnR Transcytosis.
Transcytosis of the empty TfnR was monitored essentially as described (Burgos et al., 2004). PTR or parental MDCK cells grown on 24-mm filters were treated (or not) with mCD for 60 min; thereafter, the basolateral surface was biotinylated in the cold using the cleavable sulfo-NHS-SS-biotin (0.1-mg/ml). After quenching of residual biotin, cells were incubated in MEM/BSA for 60 min at 37°C to promote trafficking. At the end of the incubation period, the apical surface of cells cultured on one set of filters was treated with reducing L-glutathione in the cold. After quenching of free SH-groups with 5-mg/ml iodoacetamide, cells were lysed in solution containing 1% Triton X-100. Streptavidin agarose-coated beads were used to precipitate biotinylated proteins in the cold, which were then analyzed by 10% SDS-PAGE. Biotinylated TfnRs were detected by Western blotting using the H68.4 monoclonal antibodies and the Fuji LAS 3000 machine was used to assess the protein band intensity. Augmented transcytosis results in the exposure of a larger fraction of biotinylated receptors at the apical surface, leading to their efficient stripping by the apical-selective glutathione treatment and consequently to a reduced signal contributed by biotinylated receptors.
Analysis of Cav1 Tyrosine 14 Phosphorylation
PTR-MDCK cells were grown on 24-mm filters in medium lacking fungicides. In some experiments, the cell's cholesterol levels were manipulated by treating with cyclodextrin and the levels of pY14-Cav1 in lysates were determined by quantitative Western blotting. In other experiments (see Figure 7B for details), pY14-Cav1 levels were quantified in cells exposed to mCD and internalizing ligands. At the end of each manipulation, cells were quickly washed in ice-cold phosphate-buffered saline (PBS) pH 7.4, and subsequently lysed in the cold in 100 µl of buffer containing 20 mM HEPES, pH 7.4, 125 mM NaCl, and 1% NP-40 supplemented with protease and phosphatase inhibitors. After a 10-min top speed centrifugation in the cold, the protein in each sample was quantified using the Bradford Reagent (Sigma). Equal protein levels (typically 30-µg) were loaded onto 13% SDS-PAGE, and Cav1 phosphorylated on tyrosine 14 was detected on Western blots, using the anti-pY14 Cav1 monoclonal antibodies.
Coimmunoprecipitation of Cav1 with pIgR
The protocol was basically adopted from other studies that established the coimmunoprecipitation (co-IP) conditions for Cav1 (Lu et al., 2001). To ensure the identification of interacting Cav1, we used a two-step IP protocol. In the first step, pIgR was IPed from two parallel samples under native conditions. Confluent PTR-MDCK cells cultured on two 10-cm dishes, in the absence of fungicides for 24 h, were washed with cold PBS, pH 7.4, and subsequently lysed in the cold in 1-ml RIPA buffer (50 mM Tris, pH 7.4, 135 mM NaCl, 1% [vol/vol] Triton X-100, and 60 mM octylglucoside) supplemented with protease and phosphatase inhibitors. Lysates were homogenized by 10 passages through a 21-gauge needle, and after a 30-min 12,000 x g centrifugation in the cold, the supernatants were divided into two identical fractions containing 1 mg protein each. Equal amounts of sheep anti-SC antibodies bound to protein A-Sepharose beads were introduced into the two lysates, and the mixtures were incubated for 60 min at 4°C, with end-to-end rotation. At the end of the incubation period, proteins bound to the beads were washed five times with RIPA buffer and released by boiling in 50 µl of 10% SDS. Nine hundred fifty microliters of 2.5% Triton dilution buffer (100 mM triethanolamine chloride, pH 8.6, 100 mM NaCl, 5 mM EDTA, 0.025% NaN3, 2.5% [wt/vol] Triton X-100) was added to the SDS solution. Cav1 was IPed for 16 h at 4°C from the released proteins, using specific anti-Cav1 antibodies, or irrelevant IgG coupled to protein A Sepharose. IPed pIgR and co-IPed Cav1 were detected using SC166 and rabbit anti-Cav1 antibodies, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD treatment affects the structure of endosomes. Confocal imaging of hTfnR, a common marker of endosomes, revealed no apparent structural changes in the shape and/or intracellular distribution of endosomes in mCD-treated MDCK cells (Supplementary Figure S1C). These measurements suggest that the integrity of MDCK cell monolayers and endosomal morphology subjected to manipulations in membrane cholesterol levels remained largely intact.
DIgA Binding Stimulates the Incorporation of pIgR into DRMs
We hypothesized that dIgA binding to pIgR increases the receptor's abundance in rafts. To investigate this hypothesis, we used filter cultured PTR-MDCK cells exposed to dIgA from the basolateral surface and assessed the presence of pIgR in floating DRMs, which biochemically may define cholesterol-sensitive membrane domains enriched with signaling factors (Foster et al., 2003). Western blots and their quantification are presented in Figure 1A, left and right panels, respectively. Although minimal levels of pIgR existed in DRMs before dIgA binding (Figure 1Aa), a greater quantity of the receptor shifted into DRMs upon 1 min of basolateral uptake of the ligand (Figure 1Ab). The shift to fractions with lighter buoyancy was further increased, reaching fractions 1 and 2, when dIgA was first internalized for 1 min and subsequently chased for 3 min in the absence of ligand (Figure 1Ac). However, after 1 min of ligand uptake and a 15-min chase (conditions leading to dIgA accumulation in AREs), the pIgR signal in the floating fractions diminished, but not to basal levels of untreated cells (Figure 1Ad). Cell treatment with mCD before dIgA uptake (for 1 min) eliminated the ability of dIgA to relocate its receptor into the lower density fractions (Figure 1Ae).
It was previously shown that a pIgR mutant lacking the carboxy-terminal 30 amino acids, pIgR-725t, is defective in dIgA transcytosis, and consequently most of the ligand is recycled. This region of pIgR's cytoplasmic tail was also shown to be required for dIgA-induced tyrosine kinase activation (Luton et al., 1998). In MDCK cells expressing pIgR-725t, low levels of mutant receptors occurred in DRMs, comparable to those observed for pIgR alone (Figure 1Af). Challenging these cells with dIgA for 1 min resulted in only a slight increase in DRM association of mutant receptors (Figure 1Ag). In these experiments, the flotation profiles of Cav1 and calnexin (Figure 1A, h–j) served as controls for DRM-associated and detergent-soluble proteins, respectively. Notably, consistent with previous reports (Foster et al., 2003), the distribution of Cav1 in DRMs was not susceptible to cholesterol depletion (Figure 1A, compare lanes h and i). This phenomenon may be attributed to structural properties that affect Cav1 interactions with cholesterol and DRMs.
Interestingly, the observation that pIgR is incorporated into DRMs after a brief exposure to the ligand suggests that these DRMs are located on the basolateral surface and juxtaposed endosomes. To further address this point, we investigated whether dIgA-pIgR complexes are associated with DRMs originating from the basolateral plasma membrane. To this end, dIgA was taken up continuously for 3 min from the basolateral side, and proteins at the basolateral surface were selectively biotinylated in the cold. Cells were solubilized in Triton X-100 and processed for flotation analysis, as before. A fraction of biotinylated pIgR associated with DRMs could be observed only in cells exposed to dIgA (Figure 1B). Taken together, the results so far indicate that dIgA binding to pIgR on the basolateral surface prompts receptor insertion into DRMs located on that surface. Association with DRMs may be pursued, but to a lesser extent in endosomes. We also concluded that the receptor's last 30 amino acids are required for dIgA-pIgR to be associated with DRMs.
Acute Cholesterol Depletion before Ligand Uptake Depolarizes the Postendocytic Transport of dIgA
The notion that dIgA-pIgR complexes enter DRMs at the basolateral surface/early endosomes suggests that cholesterol-sensitive mechanisms located at these compartments play a role in their trafficking after endocytosis. To address this hypothesis, we analyzed the endocytic and polarized postendocytic traffic of dIgA in cells whose plasma membrane cholesterol levels had been reduced. In these experiments, PTR-MDCK cells were treated for 15, 30, and 60 min at 37°C with mCD. The agent was washed out, and 125I-dIgA was internalized from the basolateral surface. Cell surface–associated ligands were removed and trafficking from endosomes to the basolateral and apical poles of the cell was monitored over time. MCD treatment for 15 min slightly facilitated the kinetics of dIgA transcytosis (Figure 2A, top panel), whereas basolateral recycling of the ligand remained unaffected (Figure 2A, bottom panel). In contrast, mCD treatment for 30 or 60 min significantly diminished dIgA transcytosis, which was accompanied by a corresponding increase in basolateral recycling. Treatment with CD had only a minor effect on transcytosis. When cholesterol was first depleted and subsequently replenished (+mCD+mCD/chol), dIgA transcytosis and recycling were restored almost completely to levels initially observed for untreated cells.
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; Subtil et al., 1999), shown to mediate pIgR endocytosis (Okamoto et al., 1992). Thus, it was reasonable to assume that cholesterol depletion inhibits dIgA-pIgR internalization. Indeed, mCD treatment conferred partial inhibition of dIgA internalization, but the inhibitory effect was instantly restored upon cholesterol replenishment (Figure 2B). Notably, whereas treatment with mCD for 15 min diminished dIgA endocytosis, the same treatment did not decrease the transcytosis of dIgA (Figure 2A), suggesting that the two processes are not necessarily coupled. Concurrent with this notion are findings indicating that transcytosis of biotinylated empty receptors internalized via a cholesterol-depleted plasma membrane was similar to that of untreated cells (Figure 2C, left panel), despite the profound inhibitory effect that mCD has on biotinylated pIgR internalization (Figure 2C, right panel). These findings were surprising because inhibition of endocytosis should have reduced the levels of subsequent transcytosis. However, as shown below, the kinetics of receptor transcytosis from endosomes is stimulated in response to cholesterol depletion, a phenomenon that may compensate for the reduced endocytic activity.
A Larger Fraction of the Human, Rather Than the Canine, TfnRs Occupies DRMs
Although TfnRs are canonically thought to be excluded from DRMs, recent evidence seems to support a possible link between TfnRs and lipid rafts: 1) recycling endosomes enriched with Tfn immunoisolated from MDCK cells contained caveolins and other raft components (Gagescu et al., 2000); 2) the folate receptor (a glycosylphosphatidylinositol-linked protein), which typically clusters in caveolae, has been found in endosomes containing Tfn (Mayor et al., 1998); 3) recycling endosomes rich in TfnRs were localized in proximity to centrosome-associated caveosomes (Maxfield and Yamashiro, 1987; Mundy et al., 2002); and 4) a fraction of TfnRs recruited under certain conditions into immunological synapses was reported to be associated with DRMs (Batista et al., 2004).
Prompted by these observations, we took a closer look into the possible link between hTfnRs and DRMs in polarized PTR-MDCK cells. Flotation analyses clearly revealed the association of a fraction of hTfnRs with DRMs (Figure 3A, top panel). In contrast with pIgR and dIgA, uptake of the ligand did not significantly alter the flotation profile of the receptor, whereas cell treatment with mCD considerably shifted its distribution toward denser fractions.
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15% of the total hTfnR population was associated with DRMs, only 4% of the cTfnR occupied these fractions (Figure 3A, bottom panel). Next, we analyzed the possibility that hTfnRs in DRMs originate at the basolateral surface. Briefly, the basolateral cell surface was biotinylated in the cold, solubilized in cold detergent, and immediately subjected to membrane flotation analysis. Probing with anti-hTfnR antibodies revealed a fraction of biotinylated receptors in DRMs (Figure 3B). Association with DRMs was slightly diminished upon receptor internalization for 5 min, and no detectable signal was observed when receptor endocytosis was extended for 15 min. These results suggest that, like in the case of dIgA bound to pIgR, a selected population of hTfnRs occupies DRMs on the basolateral surface, and their capacity to reside in DRMs is rapidly reduced upon internalization.
Cholesterol Depletion before Receptor/Ligand Internalization Depolarizes the Postendocytic Traffic of the Human But Not the Canine TfnR
Our next goal was to investigate whether these differences in DRM occupancy correlate with a differential response of TfnR's postendocytic traffic to acute cholesterol depletion. As expected from previous work (Rodal et al., 1999; Subtil et al., 1999), acute cholesterol depletion inhibited the endocytosis of both receptor types (Figure 4A, top panel). Internalization of 125I-hTfn was also inhibited by mCD treatment and was partially rescued upon subsequent cholesterol enrichment (Figure 4A, bottom panel). TfnR's transcytosis was examined by a biotinylation-based assay, schematically depicted in Figure 4B, top panel. About a 30% reduction in the signal contributed by biotinylated hTfnRs in mCD-treated cells was observed, indicating that 30% of the biotinylated receptor population transcytosed to the apical surface. Cholesterol enrichment (+mCD/chol) of membranes had no effect on hTfnR transcytosis. Under the same experimental conditions, biotinylated canine receptors were not missorted to the apical surface of mCD-treated cells.
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90%) of internalized ligand is recycled to the basolateral surface, whereas 5% is transcytosed to the apical domain. Clearly, acute cholesterol depletion resulted in an increase in basolateral-to-apical transcytosis and a parallel decrease in basolateral recycling of 125I-hTfn (Figure 4C). The effect was entirely reversible, because transcytosis and recycling of the ligand were restored to nearly the control values upon cholesterol replenishment. Cell treatment with CD had slightly attenuated only the kinetics of receptor recycling. These data principally support the possibility that disrupting the structure and function of rafts disturbs cholesterol-sensitive mechanisms that mediate polarized postendocytic trafficking.
Acute Cholesterol Depletion after Ligand Uptake Stimulates the Kinetics of Receptor Transcytosis
The observation that DRM association is diminished (but not eliminated) upon internalization suggested that cargo molecules might encounter a different cholesterol-sensitive environment in endosomes than in the plasma membrane. The next set of experiments was designed to examine the effects of cholesterol depletion on polarized postendocytic trafficking of cargo molecules whose internalization occurred via intact plasma membrane rafts. For that purpose, ligands were first internalized, and immediately thereafter, cells were subjected to acute cholesterol depletion. Surface-bound ligand was stripped off and polarized postendocytic transport was assessed. We observed that the rate of 125I-dIgA transcytosis was considerably enhanced in the cholesterol-depleted cells (Figure 5A, top). The final levels of dIgA recycling, although doubled in mCD-treated cells (Figure 5A, bottom), remained sufficiently low enough to preserve polarized transport of the ligand to the apical domain. A similar stimulatory effect on the kinetics of transcytosis was observed for the constitutive pathway of biotinylated pIgR (Figure 5B). As in the case of 125I-dIgA, the amount of biotinylated SC released to the basolateral medium was considerably higher in the cholesterol-depleted than in untreated cells (Figure 5B).
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R655-Y668; Casanova et al., 1991). The results presented in Figure 5C indicate that compared with untreated cells, the extent of metabolically labeled SC– released to the apical medium (contributed by biosynthetic delivery of pIgR to the apical surface) was not significantly different in mCD-treated cells. In contrast, the release of radiolabeled SC to the basolateral medium was markedly augmented in these cells. These results suggest that cholesterol depletion does not have a broad stimulatory effect on the apical release of SC. However, they do not preclude the possible effect of cholesterol depletion on the efficiency of pIgR cleavage to SC on the basolateral plasma membrane. Acute cholesterol depletion after ligand uptake had no detectable effect on basolateral recycling of 125I-hTfn (Figure 5D, bottom). However, the same treatment caused a twofold increase in hTfn transcytosis (Figure 5D, top), reinforcing the hypothesis that acute cholesterol depletion facilitates transcytosis. The fact that transcytosis was augmented kinetically in one case (pIgR) and elevated its final levels in another case (hTfnR) suggests that cholesterol-sensitive pathways utilized by these receptors may share common mechanisms that differ in at least one mechanistic step.
Cholesterol Enrichment Selectively Inhibits Transcytosis of dIgA
Because cholesterol depletion stimulates transcytosis of receptors internalized via undisturbed rafts, we reasoned that cholesterol enrichment would produce the opposite effect. Cholesterol enrichment by cell treatment with the mCD/chol complex neither affected the ability of dIgA to stimulate the association of pIgR with DRMs (Figure 6A), nor the capacity of 125I-dIgA to undergo endocytosis (Figure 6B, inset) and recycling (Figure 6B, bottom panel). It did, however, have a significant inhibitory effect on basolateral-to-apical transcytosis of 125I-dIgA (Figure 6B, top panel). Interestingly, the same treatment neither affected the trafficking of 125I-hTfn (Figure 6C), nor the transcytotic pathway of biotinylated pIgR (data not shown). The selective inhibitory effect may further signify cholesterol as a negative regulator of dIgA-pIgR transcytosis.
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CD Treatment Stimulates Cav1 Phosphorylation on Tyrosine 14). They also demonstrated that Cav1 is phosphorylated on tyrosine 14 (pY14-Cav1) by the oncogenic v-Src kinase (Li et al., 1996) and by normal cellular Src kinases (Lee et al., 2001; Kim et al., 2002), including c-Yes (Lee et al., 2001), a kinase previously shown to be involved in the stimulation of dIgA transcytosis (Luton et al., 1999). The proximity between intracellular caveolar structures and recycling endosomes (Gagescu et al., 2000; Mundy et al., 2002) led us to assume that a cross-talk between postendocytic membrane traffic pathways and intracellular Cav1 may be established in cholesterol-depleted cells. The result of this process could be the overall facilitation of signal transduction pathways relevant for stimulation of transcytosis.
Using specific anti-pY14-Cav1 antibodies, combined with quantitative Western blotting analysis, we demonstrated that Cav1 phosphorylation on tyrosine 14 is markedly augmented in mCD-treated cells, whereas subsequent cholesterol replenishment reduced the phosphorylation of Cav1 to nearly baseline levels. Treatment with CD had no effect, however (Figure 7A). These results indicate that Cav1 phosphorylation on tyrosine 14 responds reversibly to acute alterations in cellular cholesterol levels. Interestingly, phosphorylation levels of this residue were further elevated when cells had first been allowed to internalize dIgA and were subsequently treated with mCD (Figure 7B; +dIgA+mCD). When mCD treatment preceded ligand internalization (+mCD+dIgA), phosphorylation levels of Cav1 resembled those observed in cells treated only with mCD. Internalization of hTfn and subsequent cholesterol depletion did not appreciably stimulate Cav1 phosphorylation to levels that are higher than those observed for mCD treatment alone (+hTfn+mCD). Ligands internalized into untreated cells (+dIgA or +hTfn) had only a minor stimulatory effect on Cav1 phosphorylation.
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). These morphological data might be supported by the flotation analysis indicating that when dIgA was internalized and subsequently chased for 15 min, receptors' flotation was reduced to levels that are higher than those observed for untreated cells (see Figure 1A). Conceivably, the residual flotation quantity of pIgR could be contributed by its interactions with Cav1 in apical endosomes.
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) and Rab 25 (Casanova et al., 1999), but are relatively depleted of Tfn (Apodaca et al., 1994). Results presented in Figure 8, A and B, fulfilled these criteria, hence suggesting that Cav1-positive organelles are either linked or reside in proximity to AREs (Bush et al., 2006). Cav1 could be specifically coimmunoprecipitated (co-IPed) with anti-pIgR antibodies from cell lysates (Figure 8C), providing further biochemical support for a possible link between pIgR and Cav1 in polarized cells. Interestingly, Cav1 in the apical regions could also be localized adjacent to transcytotic dIgA in mCD-treated cells (Figure 8D), suggesting that the morphological identity of these organelles was not impaired by cholesterol depletion.
MCD Treatment Stimulates the Apical Localization of pY14-Cav1
We next analyzed the intracellular distribution of phosphorylated Cav1. In untreated cells, confocal image analysis revealed pY14-Cav1 labeling mostly in optical sections encompassing the basal and lateral regions of the cells. pY14-Cav1 was detected as diffuse staining patterns occupying the cell apex of only 10% of cells visualized in five arbitrary chosen optical sections (a representative image is shown in Figure 9A, top panel). In contrast, similar analysis of pY14-Cav1 in mCD-treated cells strikingly unraveled significant labeling of the phosphorylated protein in condensed foci located in the most apical region of 30% of the cell population (Figure 9A, bottom panel). Immunostaining of dIgA and pY14-Cav1 showed partial overlap in the apical regions of the polarized cells (Figure 9B, top panel). More specifically, for the cells presented in Figure 9B, top panel, the number of pY14-Cav1 objects whose center of mass falls within the area labeled by dIgA is an average of 1.5 SDs above the expected value for a random binomial distribution.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Hansen et al. showed that a fraction of dIgA and pIgR in membranes prepared from mouse small intestinal explants is present in DRMs. However, other studies on MDCK cells expressing pIgR reached the opposite conclusion (Sarnataro et al., 2000). Consistent with our data, Sarnataro et al. indeed showed that pIgR alone, heterologously expressed in MDCK cells, did not float to the top of density gradients. However, in contrast to data presented herein (Figure 1), they demonstrated that basolateral application of dIgA did not increase the insolubility of pIgR in cold buffer containing detergent. The discrepancy between our results and those of Sarnataro et al. (2000) most likely lies in the different experimental conditions used. First, Sarnataro et al. (2000) did not specify the filter dimensions used in their flotation analyses. We found that, owing to the relatively low abundance of pIgR in DRMs, floating pIgR was detected reproducibly only when cells were cultured on the large (75 mm) filters. Second, Sarnataro et al. (2000) applied dIgA at 1 µg/ml for 2 h before performing the assay. We found that pIgR flotation was optimal when cells were allowed to internalize 300 µg/ml dIgA for up to 3 min at 37°C. Ligand uptake followed by prolonged chase times (e.g., 15 min) led to a decrease of pIgR in DRMs (Figure 1Ad). Hence, a significant finding of our work suggests that the stimulation of pIgR flotation is an early and transient event. Lengthened incubation times with the ligand, for example, such as those applied by Sarnataro et al., may have shifted the steady-state distribution of pIgR into detergent soluble membrane compartments.
Our data are consistent with the view that cholesterol-sensitive DRMs on the basolateral surface (and/or basolateral early endosomes) harbor signaling platforms that control polarized trafficking of dIgA after endocytosis. These observations may not be entirely surprising in light of previous findings showing that DRMs are enriched with a variety of signaling molecules, including regulators of dIgA transcytosis such as the p62Yes tyrosine kinase (Luton et al., 1999; Foster et al., 2003). The possible link between DRMs and dIgA-mediated stimulation of signal transduction and transcytosis may be further emphasized by the inability of dIgA bound to pIgR-725t to induce mutant receptors to be incorporated into DRMs (Figure 1A) as well as to undergo transcytosis (Luton et al., 1998). In addition, the empty pIgR is excluded from DRMs, and its transcytosis is largely unaffected by cholesterol depletion (Figure 2C). Surprisingly, these principles were not limited to the transcytotic pIgR because a subset of the basolaterally recycled hTfnR was also found in basolateral DRMs (Figure 3), and the fidelity of its polarized postendocytic traffic was susceptible to cholesterol depletion (Figure 4). An intriguing possibility is that the hTfnR communicates with signaling molecules in rafts that facilitate its polarized trafficking to the basolateral surface after endocytosis.
It is possible that the association of pIgR (bound to dIgA) or hTfnR with DRMs is accomplished by transmembrane domains interacting with certain classes of lipids enriched in lipid rafts (Scheiffele et al., 1997; Shvartsman et al., 2003). The size of these domains could be increased in response to protein oligomerization (Harder et al., 1998). On the basis of these presumptions, dIgA-mediated dimerization of pIgR (Singer and Mostov, 1998) could have increased interactions of pIgR with DRMs. hTfnR is a homodimeric transmembrane protein (Lawrence et al., 1999), acylated on cytoplasmic cysteines (Adam et al., 1984; Jing and Trowbridge, 1987; Nadler et al., 1994). Protein acylation could play a pivotal role in targeting to DRMs (Melkonian et al., 1999); hence, in the case of the hTfnR, dimerization and acylation combined could facilitate its association with basolateral DRMs.
When cholesterol depletion was carried out after dIgA internalization, transcytosis was usually augmented without affecting the general vectorial transport. Hence, different cholesterol-dependent mechanisms may regulate the postendocytic membrane transport of cargo molecules internalized via intact basolateral lipid rafts. Caveolae are thought to play a pivotal role in maintaining the cellular cholesterol balance (Ikonen and Parton, 2000; Martin and Parton, 2005). Previous studies have shown that Cav1 is able to recognize, recruit into caveolae, and regulate the activity of proteins involved in signal transduction, among which are heterotrimeric G-proteins and Src kinases (Okamoto et al., 1998). Because some of these signaling molecules regulate transcytosis of pIgR (Bomsel and Mostov, 1992; Luton et al., 1999), we reasoned that mCD-treatment augments transcytosis by activating Cav1-associated signaling pathways. Particularly interesting in this context is the observation that phosphorylation of Cav1 on tyrosine 14 is markedly augmented in cholesterol-depleted cells (see Figure 7 and Kim et al., 2002). This signaling event, initially shown to be activated by Src kinases, can induce aggregation, fusion, and even the biogenesis of caveolar transport vesicles (Orlichenko et al., 2006). Therefore, phosphorylated Cav1 in cholesterol-depleted cells could potentially facilitate multiple membrane transport processes, among which are those that regulate transcytosis.
We suggest that mCD treatment in some way enhances cross-communication between membrane platforms containing activated Cav1 and dIgA-pIgR in a subapical endosomal compartment that possibly represent AREs. Two sets of data could agree with this interpretation. First, the abundance of pY14-Cav1 in subapical organelles increased in response to mCD treatment (Figure 9A). Second, partial colocalization between pY14-Cav1 and dIgA was evident only in optical sections taken from the very apical cytoplasm (Figure 9B). Ligand transcytosis could be facilitated by stimulation of Cav-1 dependent cargo shuttling from AREs to the apical surface. MCD-activated processes could also facilitate shuttling of dIgA from basolateral endosomes to pY14-Cav1 decorated AREs. The latter model may agree with a recent suggestion that cholesterol and glycosphingolipids are required for the delivery of transcytotic cargo from basolateral early endosomes to the subapical endosomal compartment in hepatocytes (Nyasae et al., 2003). Either event will increase the coalescence of Cav1 and dIgA-pIgR associated signaling platforms, contributing extra signaling cascades that would eventually augment both, kinase-dependent Cav1 phosphorylation and transcytosis. Clearly, the validity of these predictions warrants further investigation.
In summary, our studies suggest a bipartite model for describing the involvement of cholesterol in polarized postendocytic trafficking. One level of regulation may involve cholesterol-sensitive sorting machineries associated with DRMs at the basolateral surface and a second level of regulation may occur after endocytosis into Cav1 endosomes that modulate the efficiency of membrane transcytosis. We also provide initial evidence suggesting that the efficiency of transcytosis is fine-tuned by changes in plasma membrane cholesterol levels. Acute cholesterol depletion may have a universal impact on the stimulation of transcytosis, exemplified here for pIgR and hTfnR, and previously for the raft-Cav1–associated (Graf et al., 1999) scavenger receptor class B type I in MDCK cells (Burgos et al., 2004). Acute cholesterol enrichment yields the opposite effect.
Finally, it is well recognized that polarized epithelial cells of multicellular organisms tackle the external environment with a highly specialized apical cell membrane that differs in composition and function from the basolateral surface facing the internal milieu (Mostov et al., 1992). The former surface is highly enriched with cholesterol. Cholesterol is mostly delivered to the plasma membrane, including the apical membranes, by nonvesicular pathways that do not require passage through the Golgi apparatus (Hao et al., 2002; Maxfield and Wustner, 2002; Wustner et al., 2002). However, a significant portion of intracellular cholesterol is localized in endocytic recycling compartments, and this pool might be important for maintaining cellular cholesterol homeostasis in the plasma membrane and communicating organelles (Hao et al., 2002). We propose that the mobility of some of these cholesterol pools toward the plasma membrane could be mediated primarily by facilitating transcytosis, thereby compensating for the loss of cholesterol on the cell surface. This compensatory activity could be essential for rescuing some of the specialized epithelial-specific functions associated mostly with the apical domain.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Benjamin Aroeti (aroeti{at}cc.huji.ac.il).
Abbreviations used: DIgA, dimeric IgA; pIgR, polymeric immunoglobulin receptor; MDCK, Madin Darby canine kidney; ZO1, zonula occludens-1; DRMs, detergent-resistant membranes; mCD, methyl- -cyclodextrin; mCD/chol, mCD saturated with cholesterol; Cav1, caveolin-1; SC, secretory component; AREs, apical recycling endosomes; hTfn, human transferrin; hTfnR, human transferrin receptor; pY14-Cav1, Cav1 phosphorylated on Tyr14; IP, immunoprecipitation.
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