Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins
2007; Springer Nature; Volume: 26; Issue: 16 Linguagem: Inglês
10.1038/sj.emboj.7601813
ISSN1460-2075
AutoresKerry O. Cresawn, Beth A. Potter, Asli Oztan, Christopher J. Guerriero, Gudrun Ihrke, James R. Goldenring, Gerard Apodaca, Ora A. Weisz,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoArticle2 August 2007free access Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins Kerry O Cresawn Kerry O Cresawn Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USAThese authors equally contributed to this work Search for more papers by this author Beth A Potter Beth A Potter Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USAThese authors equally contributed to this work Search for more papers by this author Asli Oztan Asli Oztan Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Christopher J Guerriero Christopher J Guerriero Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Gudrun Ihrke Gudrun Ihrke Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Search for more papers by this author James R Goldenring James R Goldenring Department of Surgery, Vanderbilt University School of Medicine and Nashville Veterans Affairs Medical Center, Nashville, TN, USA Search for more papers by this author Gerard Apodaca Gerard Apodaca Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Ora A Weisz Corresponding Author Ora A Weisz Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Kerry O Cresawn Kerry O Cresawn Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USAThese authors equally contributed to this work Search for more papers by this author Beth A Potter Beth A Potter Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USAThese authors equally contributed to this work Search for more papers by this author Asli Oztan Asli Oztan Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Christopher J Guerriero Christopher J Guerriero Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Gudrun Ihrke Gudrun Ihrke Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Search for more papers by this author James R Goldenring James R Goldenring Department of Surgery, Vanderbilt University School of Medicine and Nashville Veterans Affairs Medical Center, Nashville, TN, USA Search for more papers by this author Gerard Apodaca Gerard Apodaca Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Ora A Weisz Corresponding Author Ora A Weisz Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Author Information Kerry O Cresawn1, Beth A Potter1, Asli Oztan1, Christopher J Guerriero1, Gudrun Ihrke3, James R Goldenring4, Gerard Apodaca1,2 and Ora A Weisz 1,2 1Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA, USA 2Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA 3Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA 4Department of Surgery, Vanderbilt University School of Medicine and Nashville Veterans Affairs Medical Center, Nashville, TN, USA *Corresponding author. Renal-Electrolyte Division, University of Pittsburgh, 3550 Terrace St., Pittsburgh, PA 15261, USA. Tel.: +1 412 383 8891; Fax: +1 412 383 8956; E-mail: [email protected] The EMBO Journal (2007)26:3737-3748https://doi.org/10.1038/sj.emboj.7601813 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Newly synthesized basolateral markers can traverse recycling endosomes en route to the surface of Madin–Darby canine kidney cells; however, the routes used by apical proteins are less clear. Here, we functionally inactivated subsets of endocytic compartments and examined the effect on surface delivery of the basolateral marker vesicular stomatitis virus glycoprotein (VSV-G), the raft-associated apical marker influenza hemagglutinin (HA), and the non-raft-associated protein endolyn. Inactivation of transferrin-positive endosomes after internalization of horseradish peroxidase (HRP)-containing conjugates inhibited VSV-G delivery, but did not disrupt apical delivery. In contrast, inhibition of protein export from apical recycling endosomes upon expression of dominant-negative constructs of myosin Vb or Sec15 selectively perturbed apical delivery of endolyn. Ablation of apical endocytic components accessible to HRP-conjugated wheat germ agglutinin (WGA) disrupted delivery of HA but not endolyn. However, delivery of glycosylphosphatidylinositol-anchored endolyn was inhibited by >50% under these conditions, suggesting that the biosynthetic itinerary of a protein is dependent on its targeting mechanism. Our studies demonstrate that apical and basolateral proteins traverse distinct endocytic intermediates en route to the cell surface, and that multiple routes exist for delivery of newly synthesized apical proteins. Introduction The generation and maintenance of the apical and basolateral surfaces in polarized epithelial cells requires efficient sorting and proper trafficking of proteins along the biosynthetic and postendocytic pathways. Protein sorting mechanisms rely on the recognition of sorting signals inherent within proteins. Basolateral sorting signals generally comprise linear peptide sequences within the cytoplasmically disposed regions of proteins, some of which fit consensus binding motifs for recognition by adaptor protein complexes (Rodriguez-Boulan et al, 2005). In contrast, apical sorting has been shown to depend on a wide variety of signals, including cytoplasmic peptide motifs, protein association with glycolipid-enriched, detergent-resistant microdomains (herein referred to as lipid rafts) through lipid anchors or transmembrane residues, and both N-and O-glycans (Rodriguez-Boulan et al, 2005; Ellis et al, 2006). Recognition of these sorting signals is thought to occur in the trans-Golgi network (TGN) based on morphological and biochemical studies demonstrating the segregation of TGN-staged apical and basolateral cargo into discrete vesicles (Wandinger-Ness et al, 1990; Keller et al, 2001; Kreitzer et al, 2003). Although the conventional model for biosynthetic protein sorting posited that post-Golgi vesicles are delivered directly to the plasma membrane, several studies over the past several years have suggested instead that some cargo traffics to the surface via endocytic intermediates (Futter et al, 1995; Leitinger et al, 1995; Orzech et al, 2000; Ang et al, 2004). Several proteins have been shown to traffic to the surface via endocytic compartments in nonpolarized cells. Results from density shift assays and endosome immunoisolation experiments, respectively, suggest that newly synthesized transferrin (Tf) receptor (Futter et al, 1995) and the asialoglycoprotein receptor (Leitinger et al, 1995) transit endocytic compartments prior to surface delivery. More recently, a temperature-sensitive mutant of the vesicular stomatitis virus glycoprotein (VSV-G) conjugated to yellow fluorescent protein (tsVSV-G-YFP) was shown by Ang et al (2004) to transit Tf-positive recycling endosomes before delivery to the surface. The indirect trafficking of tsVSV-G-YFP appears necessary for proper delivery, as inactivation of Tf-positive recycling endosomes inhibited subsequent delivery of tsVSV-G-YFP to the surface (Ang et al, 2004). Similarly, Lock and Stow (2005) used live cell imaging techniques to demonstrate trafficking of pre-staged E-cadherin from the TGN to Rab11-positive recycling endosomes of HeLa cells. Endocytic recycling compartments have also been implicated in biosynthetic trafficking in polarized cells; however, the organization of endocytic compartments is more complex in these cells. There are distinct populations of apical and basolateral early endosomes, a common recycling endosome (CRE) accessible to both apically and basolaterally internalized cargo, and a Rab11-positive apical recycling endosome (ARE). The CRE plays an important role in segregating apical and basolateral cargo, whereas the ARE is primarily involved in regulation of apically directed traffic (Hoekstra et al, 2004). The ARE has been shown to be physically and functionally distinct from other apical endocytic compartments and is typically characterized by the presence of rab11a and immunoglobulin A (IgA) and the absence of Tf (Apodaca et al, 1994; Barroso and Sztul, 1994; Gibson et al, 1998; Brown et al, 2000). A significant fraction of newly synthesized, basolaterally destined polymeric immunoglobulin receptor (pIgR) was shown to traverse the Tf-positive CRE before surface delivery (Orzech et al, 2000), suggesting that basolateral proteins transit recycling endosomes in polarized as well as nonpolarized cells. However, it remains unclear whether apical proteins also traverse endocytic compartments along the biosynthetic pathway. In previous studies, missorted apically targeted mutants of VSV-G and pIgR were suggested to transit endocytic compartments en route to the cell surface; however, the ‘apical’ variants used in these experiments possess basolateral sorting motifs that could be recognized by basolateral sorting machinery (Orzech et al, 2000; Ang et al, 2004). Additionally, there is evidence that apical proteins with different targeting signals are delivered to the surface in distinct populations of transport carriers (Jacob and Naim, 2001; Polishchuk et al, 2004); thus, there may be multiple biosynthetic routes to the apical surface. Consistent with this idea, several studies have demonstrated selective involvement of actin-dependent trafficking mechanisms in the transport of lipid-raft associated apical proteins (Jacob et al, 2003; Heine et al, 2005; Guerriero et al, 2006). To address the involvement of endocytic compartments in apical trafficking, we have selectively perturbed the function of subsets of endocytic compartments and examined the consequences on surface delivery of two proteins: the non-raft-associated protein endolyn and raft-associated hemagglutinin (HA). Endolyn is a sialomucin that efficiently traffics to the apical surface from the TGN of polarized Madin–Darby canine kidney (MDCK) cells, and subsequently cycles constitutively between the surface and lysosomes (Ihrke et al, 2001, 2004; Potter et al, 2006). Our previous studies have revealed that biosynthetic and postendocytic apical delivery of endolyn is dependent on the terminal processing of a subset of N-glycans within its lumenal domain (Potter et al, 2004, 2006). In contrast, apical delivery of HA is dependent on sorting information contained within the single transmembrane domain of this protein (Lin et al, 1998). Our results suggest that newly synthesized apical and basolateral proteins access different populations of endosomes after leaving the TGN, and moreover, that raft-associated and raft-independent proteins take different routes via distinct endocytic intermediates to the apical surface. Results Apically destined proteins do not traverse the CRE en route to the cell surface As described above, recent studies using a temperature-sensitive variant of VSV-G have demonstrated that this protein enters Tf-positive recycling endosomes before surface delivery in MDCK cells (Ang et al, 2004). This study relied on immunofluorescence, electron microscopy, and immunoisolation approaches made possible by the ability to accumulate significant levels of tsVSV-G-YFP in the endoplasmic reticulum by incubation at the nonpermissive temperature, 40°C. In compelling experiments, Tf conjugated to horseradish peroxidase (HRP) was internalized into recycling endosomes and the cells were then treated with diaminobenzidine (DAB) and hydrogen peroxide to form an insoluble precipitate that prevented fusion of post-Golgi vesicles with this compartment and consequently inhibited surface delivery of tsVSV-G-YFP. Because analogous temperature-sensitive variants are not available for most apical markers, we first confirmed the immunofluorescence results of Ang et al (2004) using subconfluent MDCK cells, and then developed a biochemical approach that would enable us to quantitate the effects of endosome ablation on surface delivery of radiolabeled apical and basolaterally directed biosynthetic cargo in fully polarized MDCK cells. MDCK cells that stably express human Tf receptor (TfR; PTR9 cells) were grown on coverslips and infected with replication-deficient adenovirus encoding tsVSV-G-YFP and incubated at 40°C overnight to accumulate the protein in the endoplasmic reticulum. The next day, HRP-Tf was internalized basolaterally for 45 min at 40°C to allow accumulation in recycling endosomes. To inactivate recycling endosomes, cells were incubated with DAB and 0.025% hydrogen peroxide for 1 h on ice. As additional controls, we omitted either the HRP-Tf incubation (no HRP-Tf) or the hydrogen peroxide (no H2O2) in these experiments. After a 1-h chase at the permissive temperature of 32°C in the presence of cycloheximide to prevent new protein synthesis, the cells were fixed and imaged (Figure 1A). In concordance with the observations of Ang et al (2004), delivery of tsVSV-G-YFP to the surface was only inhibited when recycling endosomes were successfully inactivated by incubation with both DAB and hydrogen peroxide. Figure 1.Inactivation of Tf-containing endosomes in nonpolarized and polarized cells inhibits surface delivery of tsVSV-G-YFP. (A) PTR9 cells grown on coverslips were infected with adenovirus-encoding tsVSV-G-YFP and incubated overnight at 40°C. The next day, the cells were incubated in serum-free media for 30 min before addition of HRP-Tf for 45 min at 40°C. Remaining surface HRP-Tf was stripped and Tf-containing endosomes were inactivated on ice as described in Materials and methods. As controls in this and subsequent experiments, cells were incubated either without HRP-Tf, DAB, or H2O2 (no add'n), with only HRP-Tf and DAB (no H2O2), or with only DAB and H2O2 (no HRP-Tf). Cells were then incubated at 32°C in the presence of cycloheximide for 1 h, washed with PBS, then fixed and imaged. Scale bar=5 μm (B) Polarized PTR9 cells grown on filters were infected with adenovirus encoding VSV-G and incubated overnight at 37°C. The following day, cells were starved in medium devoid of cysteine and methionine for 30 min and then radiolabeled for 15 min. HRP-Tf was included in the basolateral medium during the starve and radiolabeling period. After stripping residual surface HRP-Tf, cells were treated with or without DAB and H2O2 for 1 h on ice as in (A). After washing, cells were chased for 0 or 90 min at 37°C, and delivery to the basolateral surface was quantified using domain-selective biotinylation. Samples were immunoprecipitated and analyzed by SDS–PAGE and gels showing the total amount of radiolabeled protein in the cell and the amount at the basolateral surface in a representative experiment are shown (upper panel). The mean±s.e. of three experiments is plotted (lower panel). Surface delivery of VSV-G was significantly inhibited in the ‘complete’ reaction samples compared with all three control conditions (*P<0.02 by Student's t-test). Download figure Download PowerPoint We next modified this protocol to enable quantitation of the effects of inactivating Tf-positive CRE on basolateral delivery of radiolabeled VSV-G in fully polarized cells. These and our other biochemical experiments were variously performed using adenoviruses encoding either tsVSV-G-YFP or wild-type VSV-G; and because we obtained indistinguishable results with both, the data have been combined. In the description of these experiments, the term VSV-G thus refers to both constructs. Virally-infected filter-grown PTR9 cells were radiolabeled for 15 min in the presence or absence of HRP-Tf and the cells were incubated with or without DAB and hydrogen peroxide for 1 h on ice. Cells were then chased at 37°C (for VSV-G) or 32°C (for tsVSV-G-YFP) for 0 or 90 min, after which the basolateral surface was biotinylated. A representative gel from one experiment is shown in Figure 1B. Consistent with our immunofluorescence results in Figure 1A, quantitation of the results from several biochemical experiments confirmed that inactivation of HRP-Tf containing endosomes significantly inhibited basolateral delivery of VSV-G by about 50%. VSV-G was not missorted to the apical surface under these conditions, as CRE inactivation had no effect on the total amount of VSV-G delivered to the apical plasma membrane (KO Cresawn and BA Potter, unpublished data, 2007). Having validated our biochemical approach to examine the itinerary of VSV-G, we next determined the effect of inactivating the CRE on apical trafficking of endolyn and HA. These experiments were performed essentially as described for Figure 1B, except that apical delivery of radiolabeled HA was quantitated using a cell surface trypsinization assay. Strikingly, inactivation of the CRE did not significantly decrease the amount of newly synthesized endolyn (Figure 2A) or HA (Figure 2B) that reached the apical surface, suggesting that these proteins do not traverse the CRE along the biosynthetic pathway. In these experiments, we routinely observed that a population of immature (nonsialylated) protein was recovered in the biotinylated fraction of all samples treated with hydrogen peroxide, suggesting that terminal glycosylation is perturbed by this reagent (compare surface 90 min no HRP-Tf/complete lanes with no add'n lanes). This was also the case for VSV-G (Figure 1B); however, the electrophoretic mobility shift is less obvious as this protein has only two N-linked glycans. Regardless, the aberrant glycosylation did not apparently perturb the efficiency of surface delivery, as surface delivery of fully and aberrantly processed endolyn was similar when the two fractions were quantitated separately (data not shown). This was somewhat surprising as we have previously shown that terminal glycosylation of endolyn N-glycans is required for polarized apical delivery (Potter et al, 2004). We hypothesize that peroxide may not affect the key determinant required for apical targeting, or alternatively, because only a fraction of endolyn is affected, multimerization or other mechanisms may compensate to enable efficient delivery of the entire pool of newly synthesized proteins. Figure 2.Delivery of endolyn and HA to the apical surface is not disrupted when Tf-containing endosomes are inactivated. Filter-grown PTR9 cells were infected with adenoviruses encoding endolyn (A) or HA (B) and incubated overnight at 37°C. Cells were radiolabeled, incubated with HRP-Tf at 37°C, and subjected to crosslinking as described for Figure 1B. Apical delivery was quantified by domain-selective biotinylation for endolyn (M=mature, I=immature) and by cell surface trypsinization for HA (resulting in two cleaved products marked HA1 and HA2). Uncleaved (intracellular) HA is marked as HA0. Samples were analyzed by SDS–PAGE and the gels from a representative experiment for each marker are shown (left panels). The percent apical delivery (mean±s.e.m.) for endolyn (n=3) and HA (n=3–6) are shown (right panels). Surface delivery of HA and endolyn in the ‘complete’ reaction samples was statistically indistinguishable from all three control conditions. Download figure Download PowerPoint Apical protein delivery is differentially sensitive to perturbation of ARE function Given the segregation of recycling compartments in polarized cells, we next asked whether endolyn and HA traverse the ARE along the biosynthetic pathway. Attempts to generate a functional HRP-IgA conjugate to inactivate this compartment were unsuccessful. As an alternative, we examined the apical trafficking of endolyn and HA in MDCK cells stably expressing an inducible GFP-tagged myosin Vb tail fragment (GFP-MyoVbT) which acts as a dominant-negative inhibitor of membrane traffic out of the ARE (MyoVbT cells; Lapierre et al, 2001). This construct can interact with Rab11, but lacks a motor domain, and has previously been shown to cause the accumulation of transcytosing and apical recycling cargo in the ARE (Lapierre et al, 2001). To confirm this phenotype, we examined by immunofluorescence microscopy whether IgA internalized from the basolateral surface for 10 min and chased for 1 h accumulates in a GFP-positive subapical compartment as previously reported (Lapierre et al, 2001). Confocal images of optical sections through the subapical region reveal an accumulation of IgA in the ARE of MyoVbT cells not seen in parental MDCK cells (Figure 3A, panels b and d, respectively). Additionally, basolateral to apical transcytosis of pre-internalized 125IgA was reduced by 35% after 1 h in MyoVbT cells compared with control cells, consistent with a perturbation of trafficking from the ARE in these cells (CJG, unpublished data, 2006). Figure 3.Biosynthetic apical delivery of endolyn, but not HA, is disrupted in cells expressing the myosin Vb tail. (A) IgA (200 μg/ml) was internalized from the basolateral surface of filter-grown MyoVbT or control MDCK cells for 10 min and chased for 60 min at 37°C. After rinsing, the cells were fixed, permeabilized, and incubated with Cy-5-conjugated anti-human IgA. Confocal sections taken just beneath the apical pole of the cell are shown; panel a: GFP-MyoVbT; panel b: IgA; panel c: overlay of a and b; panel d: IgA in control MDCK cells. Scale bar=10 μm. (B–D) Filter-grown MyoVbT or control cells were infected with adenoviruses encoding endolyn (B), HA (C), or VSV-G (D). The following day, cells were starved, radiolabeled for 15 min, and chased for the indicated periods at 37°C before quantitation of surface delivery. The percent surface delivery is plotted as the mean±range of two experiments with duplicate samples for endolyn and HA and four experiments for VSV G. *P<0.001 compared with control cells. Download figure Download PowerPoint To examine the effect of perturbing ARE function on the apical delivery of endolyn and HA, MyoVbT or control cells were infected with recombinant adenovirus expressing either construct. Cells were radiolabeled for 15 min and subsequent delivery of endolyn or HA to the apical surface was assessed upon warming the cells for 0, 30, 60, or 90 min at 37°C. As shown in Figure 3B, apical delivery of endolyn was significantly inhibited in MyoVbT cells compared to controls. Inhibition was observed even at the earliest time point, suggesting that biosynthetic rather than postendocytic delivery of endolyn was disrupted. In contrast, apical delivery kinetics of HA were comparable in both parental and MyoVbT cell lines (Figure 3C). Similarly, there was no effect on the kinetics of basolateral delivery of VSV-G in MyoVbT cells (Figure 3D), suggesting that of these three cargoes, only endolyn traverses the ARE en route to the surface. As a complementary approach, we examined the effect of another perturbant of Rab11 function on apical delivery kinetics of endolyn and HA. Two hybrid analysis using Drosophila and canine homologs have revealed an interaction between Rab11a and the C-terminal domain of the Sec15 subunit of the exocyst (Zhang et al, 2004; Wu et al, 2005) and recent studies in MDCK cells have implicated a functional role for this interaction in IgA transcytosis (Oztan et al, in press). In these studies, expression of a C-terminal construct of Sec15 fused to GFP (Sec15CT) in polarized MDCK cells resulted in the redistribution of Rab11a and slowed transcytosis kinetics, but did not alter apical recycling of IgA. In contrast, expression of a GFP-tagged Sec15CT construct containing a point mutation that disrupts the interaction of the protein with Rab11 [Sec15CT(NA), in which Asn709 is replaced by alanine] did not inhibit transcytosis (Oztan et al, in press). For our experiments, MDCK cell lines stably expressing Sec15CT or Sec15CT(NA) were infected with adenovirus-expressing endolyn or HA and apical delivery of the proteins was quantitated as described above. Apical transport kinetics of endolyn were inhibited in cells expressing Sec15CT compared with parental cells and cells stably expressing the Sec15CT(NA) mutant (Figure 4A). In contrast, HA delivery was unaffected by expression of either Sec15CT construct (Figure 4B). Figure 4.Expression of a dominant-negative inhibitor of Sec15 selectively disrupts apical delivery of endolyn. MDCK cells stably expressing Sec15 or Sec15(NA) and parental cells were co-infected with adenovirus-expressing transactivator and either endolyn (A) or HA (B). The following day, cells were radiolabeled for 15 min and chased from 0 to 90 min at 37°C before quantitation of apical delivery. The percent surface delivery is plotted as the mean±s.e. of three experiments for endolyn and two for HA. *P<0.05 compared with control cells. Download figure Download PowerPoint We used indirect immunofluorescence microscopy as a third approach to examine whether newly synthesized endolyn traverses a MyoVbT-/Rab11a-positive compartment en route to the apical surface. Endolyn was briefly expressed in MyoVbT cells via adenovirus and then incubated at 19°C for 2 h to accumulate newly synthesized biosynthetic cargo in the TGN. Cells were then warmed to 37°C for 20 min, fixed, incubated with antibodies to detect endolyn and Rab11a, and processed for confocal microscopy (Figure 5). Before warm-up, endolyn and rab11a were localized to distinct nonoverlapping compartments (data not shown). However, after warming for 20 min, significant colocalization between endolyn, GFP-MyoVbT, and Rab11a was observed (Figure 5C, E, and F). In contrast, no colocalization between HA staged in a similar fashion and GFP-MyoVbT was observed (Supplementary Figure 1). To ensure that the colocalization between endolyn and MyoVbT did not reflect postendocytic trafficking of endolyn that had reached the apical surface, some filters were incubated with anti-endolyn antibody during the last hour of the 19°C stage, then washed and incubated at 37°C for 20 min to track the route of any endolyn already at the surface before warming (Figure 5H). Internalized anti-endolyn antibody did not colocalize with GFP-MyoVbT, suggesting that endolyn recycles primarily from other apical endocytic compartments. Colocalization between post-TGN endolyn and rab11a was also observed in parental cells (Supplementary Figure 2). Together with the results from the MyoVbT and Sec15CT cell experiments, these data suggest that there are differential requirements for endocytic compartments in the delivery of subsets of apical proteins along the biosynthetic pathway, and point to a selective involvement of the ARE in apical biosynthetic trafficking of endolyn but not HA. Figure 5.Newly synthesized endolyn colocalizes with MyoVbT and rab11a. Filter-grown MyoVbT cells were infected with adenovirus-expressing endolyn, incubated for 6 h at 37°C to initiate endolyn synthesis, and then moved to 19°C for 2 h to accumulate newly synthesized proteins in the TGN. Cells were then warmed to 37°C for 20 min to release staged proteins, then fixed and processed for detection of GFP-MyoVbT (green (B)), endolyn (red (A)), and rab11a (blue (D)). Individual confocal sections taken just beneath the apical pole of the cell are shown in each panel. Colocalization of GFP-MyoVbT with rab 11a is shown in (F); overlay of endolyn with GFP-MyoVbT, rab11a, and the triple overlay are shown in (C), (E), and (G), respectively. Cells in (H) were incubated with apically added monoclonal anti-endolyn antibody during the last hour at 19°C, then warmed to 37°C for 20 min to track the postendocytic itinerary of any endolyn that had reached the cell surface. Scale bar=20 μm, (A–G); 10 μm, (H). Download figure Download PowerPoint Inactivation of HRP-wheat germ agglutinin-containing compartments selectively disrupts apical delivery of HA Although the studies above suggest a role for the ARE in apical delivery of endolyn, they do not rule out the possibility that newly synthesized apical proteins traverse other apical endocytic compartments before cell surface delivery. We therefore examined whether internalization of wheat germ agglutinin (WGA) would provide access to a broader subset of apical endocytic compartments. WGA binds GlcNAc- or sialic acid-containing oligosaccharides on glycosylated membrane proteins and is efficiently internalized into the apical endocytic pathway. To characterize which compartments WGA can access when internalized from the apical surface of polarized MDCK cells, FITC-conjugated WGA was internalized apically for 15 min at 37°C. Cells were then fixed, permeabilized, and processed with antibodies for markers of various compartments: the ARE marker Rab11a; the early endosome marker EEA1; the TGN marker furin; the late endosome marker mannose-6-phosphate receptor; and the lysosomal marker Lamp2. There was extensive colocalization of internalized FITC-WGA with the early endosomal marke
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