Trafficking of galectin-3 through endosomal organelles of polarized and non-polarized cells

Trafficking of galectin-3 through endosomal organelles of polarized and non-polarized cells

European Journal of Cell Biology 89 (2010) 788–798 Contents lists available at ScienceDirect European Journal of Cell Biology journal homepage: www...

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European Journal of Cell Biology 89 (2010) 788–798

Contents lists available at ScienceDirect

European Journal of Cell Biology journal homepage: www.elsevier.de/ejcb

Trafficking of galectin-3 through endosomal organelles of polarized and non-polarized cells Dominik Schneider 1 , Christoph Greb 1 , Annett Koch 2 , Tamara Straube, Alexandra Elli, Delphine Delacour 3 , Ralf Jacob ∗ Department of Cell Biology and Cell Pathology, Philipps-Universität Marburg, Marburg, Germany

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Article history: Received 12 April 2010 Received in revised form 25 June 2010 Accepted 1 July 2010 Keywords: Galectin-3 Apical trafficking Endosomes COS-1 MDCK Sorting

a b s t r a c t In epithelial cells, the ␤-galactoside-binding lectin galectin-3 mediates the non-raft-dependent glycoprotein targeting to the apical membrane domain. In this study, we aimed to identify intracellular compartments involved in the trafficking of galectin-3. By studying fluorescent fusion proteins in living cells, we could show that galectin-3 accumulates intracellularly in acidified endosomes. Total internal reflection fluorescence microscopy studies of the apical surface of polarized MDCK cells revealed that galectin-3 is enriched in tubular and vesicular Rab11-positive recycling endosomes in the vicinity of the apical cell surface. These endosomal organelles are candidate compartments for the association between galectin-3 and exocytic apical cargo. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved.

Introduction The vast majority of secretory proteins are transported along the secretory pathway prior to secretion to the extracellular milieu (Palade, 1975). However, a small protein population utilizes alternative pathways independent of the passage through endoplasmic reticulum (ER) and Golgi apparatus (for review see Nickel and Seedorf (2008)). Among these are the so-called galectins, which are soluble, non-glycosylated ␤-galactoside binding lectins of small molecular weight between 14 and 36 kDa (Barondes et al., 1994). Galectin-3 is composed of a single C-terminal carbohydrate recognition domain (CRD) linked to a unique N-terminal domain involved in the oligomerization of the polypeptide (Nieminen et al., 2007). It is abundant in the cytosol as well as extracellularly (Sato et al., 1993) and is mainly expressed in cells of the immune system, epithelial cells and some sensory neurons (Barondes et al., 1994).

∗ Corresponding author at: Department of Cell Biology and Cell Pathology, Philipps-Universität Marburg, Robert-Koch-Str. 6, D-35033 Marburg, Germany. Tel.: +49 6421 286 6482; fax: +49 6421 286 6414. E-mail address: [email protected] (R. Jacob). 1 Both authors have contributed equally to this work. 2 Present address: Charité-Universitätsmedizin Berlin, CCM, Institut für Biochemie, Monbijoustr. 2, 10117 Berlin, Germany. 3 Present address: Institut Jacques-Monod, CNRS-UMR7592, Université Paris Diderot, 15 Rue Hélène Brion, 75013 Paris, France.

In polarized epithelial MDCK cells about 12% of galectin-3 is secreted from the apical membrane, while less than 2% was found in the basolateral medium (Lindstedt et al., 1993). In MDCK cells this relatively slow release could be blocked at an incubation temperature of 20 ◦ C and by microtubule disruption with nocodazole, two striking similarities to the classical secretion pathway. This indicates that the apical secretion of galectin-3 is directly or indirectly linked to intracellular trafficking events. Corroborating this conclusion, we could recently identify a functional role of galectin-3 in apical sorting of a subset of glycoproteins that do not associate with sphingolipid/cholesterol enriched membrane microdomains or lipid rafts (Delacour et al., 2006). Knockdown of galectin-3 in MDCK cells or knockout of this gene in mice resulted in an aberrant basolateral localization of normally apically targeted glycoproteins (Delacour et al., 2008). The lectin was identified in post-Golgi carriers for apical cargo and could be pulled down in association with apical glycoproteins in a carbohydrate dependent manner. In view of the fact that galectin-3 can oligomerize into cross-linking complexes with multivalent carbohydrates (Ahmad et al., 2004), a putative scenario of galectin-3 mediated apical sorting would imply the formation of multimeric lattices. These lattices then segregate non-raft associated apical glycoproteins into specific carriers (Delacour and Jacob, 2006). Evidence for this hypothesis comes from the observation that galectin-3 as well as apical glycoproteins could be identified in high molecular weight clusters (HMWCs) of polarized epithelial cells (Delacour et al., 2007). The galectin3 dependent HMWCs are assembled about 10 min after the newly synthesized material has left the TGN. However, compartments,

0171-9335/$ – see front matter. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2010.07.001

D. Schneider et al. / European Journal of Cell Biology 89 (2010) 788–798

where galectin-3 meets apical cargo remain to be identified (Weisz and Rodriguez-Boulan, 2009). In this study, we used fluorescence microscopic techniques to identify an endosomal accumulation of galectin-3 in non-polarized COS-1 and polarized MDCK cells. Results and discussion Endosomal localization of galectin-3 Based on our observation that galectin-3 was present within immunoisolated post-Golgi carriers we tried to identify post-Golgi compartments involved in non-classical secretion of galectin-3. Non-polarized COS-1 cells are proper to address this point since they grow on a coverslip with a flat, fried egg-like morphology. This allows the study of intracellular compartments in a single focal plane. At first, fluorescent Gal3-YFP was co-expressed in combination with trans Golgi galactosyltransferase (GT)-CFP or endosomal Rab4, -8 or -11 fused to CFP. Image stacks of CFPor YFP-fluorescence of co-transfected living cells were recorded at 37 ◦ C over time and 3D-reconstructed images are presented in Fig. 1. Gal3-YFP-fluorescence appeared as a strong fluores-

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cence in distinct vesicular structures of 0.5–2 ␮m. In Fig. 1A the absence of a significant overlay between GT-CFP and Gal3-YFP indicates that the lectin does not accumulate in the trans area of the Golgi (Fig. 1A). If we now visualize Gal3-YFP in comparison to Rab4-CFP, which plays a role in fast recycling from early endosomes back to the plasma membrane (Sheff et al., 1999; van der Sluijs et al., 1992), about one-third of the galectin-3 positive vesicles were co-labelled with the Rab-GTPase. A similar degree of co-localization was observed when the recycling endosome marker Rab11-CFP (Urbe et al., 1993) was co-transfected. Both Rab-GTPases were detected in relatively large sorting endosomes as well as in smaller vesicles as demonstrated before (Sonnichsen et al., 2000) and labelled specific endosomal domains involved in cargo recycling. This suggests that a representative population of galectin-3 accumulates in recycling endosomes. Rab8, another Rab-GTPase involved in membrane recycling (Hattula et al., 2006) and apical trafficking (Cramm-Behrens et al., 2008), also co-localizes with galectin-3 containing structures but to a lesser extent (Fig. 1A). This divergence in co-localization between Rab4/-11 or Rab8 and galectin-3 refers to distinct endosomal domains traversed by the lectin, which are more or less included in membrane recycling pathways.

Fig. 1. Transiently transfected COS-1 cells accumulate galectin-3 in acidified endosomes. COS-1 cells were transfected with galactosyltransferase -, Rab4-, Rab8- or Rab11CFP and Gal3-YFP. 48 h after transfection the cells were analyzed by fluorescence microscopy. The recorded image stacks were processed by deconvolution and background elimination. Dual colors are depicted in the 3D-reconstructed images. The Pearson correlation coefficient (P.c.c.) of each image is depicted on the right. (A) Vesicles positive for each of the fluorescent fusion protein were counted from at least three populations of three co-transfected cells for each Rab protein for quantification. A Pearson correlation coefficient of more than 0.9 was assigned to co-localization of structures (depicted in white). Error bars (±SEM) are indicated. Scale bars, 10 ␮m. Arrows indicate below the x- (green), y- (red) and z- (blue) orientation. (B) Sections of a 4D-time-lapse series from the galactosyltransferase-CFP/Gal3-YFP double transfected cell shown in (A). A vesicular structure that disappears in the series is encircled and time intervals are indicated in seconds (see also Supporting movie 1). Scale bars, 10 ␮m. (C) COS-1 cells transfected with Rab4-CFP and Gal3-YFP were analyzed by fluorescence microscopy before and after treatment with NH4 Cl (see also Supporting movie 2). Rab4-positive structures with an increase in Gal3-YFP-fluorescence following NH4 Cl addition are indicated by arrows. Scale bars, 20 ␮m. (D) Co-localized vesicular structures assigned by Pearson correlation from four individual experiments were quantified. Error bars represent the mean ± SEM. The measurements were analyzed using a Student’s t-test (**p < 0.01). (E) Gal3-pHrodo was added to the medium of COS-1 cells at 4 ◦ C. The recombinant protein was internalised for 0 or 30 min at 37 ◦ C as indicated and analyzed by fluorescence microscopy for pHrodo fluorescence. Scale bars, 10 ␮m.

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Time-lapse recording of cells expressing GT-CFP and Gal3-YFP revealed galectin-3 harbouring vesicles, which were transiently in close proximity to the trans Golgi area (Fig. 1B, Supporting movie 1). In some cases, those vesicles then moved to the periphery and Gal3-YFP-fluorescence vanished, although the whole cell has been recorded. One explanation could be that this loss of fluorescence is due to the fact that the chemical environment of Gal3-YFP has changed on this passage. The fluorescence of green fluorescent proteins strongly depends on the pH of intracellular compartments in living cells and decreases about 10-fold by a change from pH 7.4 to 6.0 (Llopis et al., 1998; Kneen et al., 1998), which resembles the pH of endosomal compartments (Rodriguez-Boulan et al., 2005). We therefore wanted to examine if Gal3-YFP could accumulate inside of acidified Rab4-positive compartments by the addition of NH4 Cl to living cells. NH4 Cl dissociates in aqueous solution into NH3 , which can penetrate cell membranes and shifts the pH by being protonated to a more basic milieu. When COS-1 cells were transfected with Rab4-CFP and Gal3-YFP some Rab4-positive vesicles were co-stained by galectin-3 as described above, while several vesicles were negative for the lectin. As indicated in Fig. 1C, significant amounts of Gal3-YFP-fluorescence appeared after the addition of NH4 Cl in Rab4-positive endosomes (see also Supporting movie 2). When COS-1 cells were now co-transfected with Gal3-YFP and the pH-insensitive Gal3-DsRedmonomer (Gal3-DsRed), vesicular structures with a constant DsRed-fluorescence showed a dramatic increase in YFP-fluorescence after NH4 Cl addition (Supplementary Fig. S1). This could be interpreted by an accumulation of galectin-3 in endosomal organelles with a more acidic pH. Further evidence for an accumulation of galectin-3 in acidified compartments came from studies with exogenously added recombinant galectin-3 fused to pHrodo (Gal3-pHrodo), a dye that is non-fluorescent at neutral pH and fluoresces bright red in acidic environments. Here, pHrodo fluorescence could be detected in COS-1 cells about 30 min after endocytic uptake (Fig. 1D). Altogether, these data suggest that cellular trafficking of galectin-3 comprises the transport through acidified endosomal compartments in living COS-1 cells. Fusion of galectin-3 positive carrier vesicles with the plasma membrane Next, we addressed the question if endosomal galectin-3 is exocytosed by vesicle fusion with the plasma membrane. Since our previous data suggest that this step interferes with exocytic traffic of newly synthesized material (Delacour et al., 2006), we first synchronized this pathway in COS-1 cells by a temperaturedependent TGN block. At 37 ◦ C, TGN-accumulated material was released for 30 min and epifluorescence imaging depicted Gal3YFP-fluorescence concentrated in vesicular structures or as diffuse cytosolic staining (Fig. 2A). To image fluorescence within or in close proximity to the plasma membrane we illuminated from the surface to a depth of less than 90 nm by total internal reflection fluorescence microscopy (TIRFM). Here, as recorded by time-lapse imaging fluorescent spots appeared superimposed on a dim glow of the plasma membrane (Fig. 2, Supporting movie 3). As monitored by linescan analysis, fluorescence intensity reached a maximum at the centre of docking followed by widening and flattening of the graph (Fig. 2C). This profile is characteristic for vesicle docking and fusion. Moreover, these spots were visible only for a few video frames as they brightened. Quantitative analysis revealed a plateau of elevated total intensities (Fig. 2D), which is most likely based on lateral spreading of Gal3-YFP according to previously monitored VSVG-GFP fusion events (Schmoranzer et al., 2000). Together, these data demonstrate that in COS-1 cells galectin-3 is transported by vesicular carriers to the plasma membrane and released by vesicle fusion into the extracellular milieu.

Accumulation of galectin-3 in recycling endosomes of MDCK cells Up to now we had used non-polarized COS-1 cells to visualize galectin-3 trafficking. Coming back to epithelial cells we now switched to MDCK cells as model system. Since it had previously been published that MDCK cells secrete galectin-3 from the apical membrane we studied galectin-3 distribution at this cell pole. Therefore MDCK cell lines stably expressing Gal3-DsRed and the apical marker p75NTR -GFP (MDCKIIp75GFP/Gal3DsRed ) or exclusively Gal3-DsRed (MDCKIIGal3DsRed ) were incubated on polyethylene terephtalate (PET) filters for at least 5 days to form a polarized cell monolayer. Filters were excised and placed top down onto a coverslip. At first, co-localization between endogenous galectin-3 and Gal3-DsRed was validated in MDCKGal3DsRed cells by immunostaining with pAb anti-Gal3. As depicted in Fig. 3 Gal3-DsRed as well as endogenous galectin-3 labelled vesicular and sometimes also tubular structures above the nucleus close to the apical membrane. Excitation by TIRF microscopy visualized similar structures, which are within the 100–300 nm distance from the coverslip and thus within or in close proximity to the apical membrane. Analogous images were acquired for the Gal3-YFP construct in MDCK cells (data not shown). In a second set of experiments with endogenously expressed galectin-3 we inspected co-localization of Rab-positive endosomes. In each case a constant but minor cohort of galectin3 was co-stained with Rab4-, Rab8- or Rab11-positive subapical vesicular structures (Fig. 4). This supports our above indicated observations in COS-1 cells and suggests that a contingent of the lectin traverses Rab4-, Rab8- and Rab11-positive endosomes in MDCK cells. With a focus on the subapical localization of galectin-3 in MDCK cells, fluorescently labelled galectin-3 was excited by TIRF at the apical cell pole. We started with the analysis of galectin-3 in comparison to p75NTR in MDCKIIp75GFP/Gal3DsRed cells. Fig. 5A depicts long tubular structures of about 2–4 ␮m length and less than 0.2 ␮m in diameter, which were positive for p75 but at least in part also stained by galectin-3. They correspond most likely to microvilli. Moreover, some vesicular and tubular structures that were predominantly stained by galectin-3 and only slightly p75positive were located in central areas of the apical domain. To display the allocation of galectin-3 and p75 in the third dimension, TIRF-images were recorded at varying penetration depths, processed by subtraction and rendered for 3D reconstruction (Fig. 5B). The resulting images depicted a concentration of p75 towards the apical membrane (see also Fig. 5G), while only minor amounts of galectin-3 stained this section. On the other hand, representative quantities of the lectin were enriched in a vesicular or tubular compartment beneath the apical membrane. Interestingly, a similar concentration gradient could be detected in MDCKp75GFP/Rab11DsRed cells, stably expressing p75-GFP and Rab11-DsRed (Fig. 5C and D). Here, Rab11 was concentrated in submembraneous tubules and vesicles similar to previously described recycling endosomal structures (Apodaca et al., 1994). A network of endocytic vesicles and vacuoles immediately beneath apical membrane invaginations has also been visualized by scanning electron microscopy (Hatae et al., 1997). This network is located within the 300 nm range from the plasma membrane and therefore in the excitation range for TIRFM of a flattened apical membrane domain. It thus seems very likely that major amounts of galectin-3 are stored in this subapical endosomal compartment prior to its release from the apical cell surface. This could be confirmed by direct co-labelling of galectin-3 and Rab11 as indicated in Fig. 5E and F. Tubular as well as vesicular structures were positive for galectin-3-YFP, while Rab11 labelled predominantly vesicles which were at least in part co-stained by the lectin. To verify that membrane-bound as well as submembraneous intracellular structures are excited by this TIRFM-setup, we compared immunostained p75 by anti-p75

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Fig. 2. Fusion of galectin-3 positive vesicles with the plasma membrane of COS-1 cells. COS-1 cells expressing Gal3-YFP were incubated for 4 h at 20 ◦ C. 30 min after the temperature block TIRFM was applied to monitor fusion events at the plasma membrane. (A) Epifluorescence as well as TIRF images are depicted and areas of fusion are indicated by circles (see also Supporting movie 3). Scale bars, 10 ␮m. (B) Six images recorded every 3 s illustrate the fusion of a vesicular structure with the plasma membrane. A corresponding graph in (C) shows the linescan intensity profile. Scale bar represents 2 ␮m. Total linescan intensities over time from five fusion events are depicted in (D).

antibodies directed against the extracellular domain with GFPfluorescence of MDCKp75-GFP cells. Fig. 5G indicates that in addition to signals from antibodies at the cell surface, p75-GFP also labels vesicular and tubular structures. Therefore, we could also monitor compartments located just beneath the apical membrane, which are inaccessible to the antibody. Similar control experiments performed with anti-galectin-3 antibodies in comparison to Gal3DsRed revealed that in conjunction with the 3D-reconstruction TIRF-images galectin-3 could be detected in an extensive network beneath the apical membrane which was inaccessible for the antibody. To conclude, these observations suggest that galectin-3 accumulates in non-polarized and polarized cells in endosomal recycling compartments. Since the apical recycling pathway of MDCK cells comprises an early compartment with a pH of about 5.8 and an apical recycling endosome (ARE) whose pH is about 6.5 (Wang et al., 2000), we now monitored the fluorescence of galectin-3-YFP

before and after neutralisation with NH4 Cl by TIRF microscopy. As depicted in Fig. 6A in some cells YFP-fluorescence appeared in relatively large structures of vesicular appearance close to the apical cell surface as a consequence of NH4 Cl treatment. These findings were corroborated by the uptake of Gal3-pHrodo from the apical surface of MDCK cells into endosomal structures. As indicated in Fig. 6B Gal3-pHrodo fluorescence rose dramatically with increasing periods of uptake, thus stressing the fact that galectin3 accumulates in acidified subapical endosomal compartments of MDCK cells. Apical uptake of galectin-3 We then studied this apical uptake process of galectin-3 from the extracellular milieu by adding exogenous lectin to the apical medium of polarized MDCK cells. Therefore, recombinant galectin-3 covalently linked to Alexa633 and dextran-Alexa488

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Fig. 3. Fluorescence microscopic analysis of Gal3-DsRed and endogenous galectin-3 in MDCK cells. MDCKIIGal3DsRed cells were grown on filters for 6 days. The cells were fixed and immunostained with anti-galectin-3/Alexa488 antibodies. Filters were excised and positioned upside down on a coverslip for epifluorescence or TIRF microscopy. Immunostaining of endogenous galectin-3 and Gal3-DsRed are depicted individually or in a merged image. Co-stained structures are indicated by arrows. 20 layers of the cells were recorded from top to bottom for 3D reconstruction. A diagonal top view on the reconstructed epithelial monolayer is depicted. Arrows indicate below the x(green), y- (red) and z- (blue) orientation. Scale bars, 10 ␮m.

were monitored by confocal fluorescence microscopy from subapical regions after 1 min of internalisation and varying intervals of chase at 37 ◦ C (Fig. 7A). Following 1 min of chase about 28 ± 6% of galectin-3 co-localized with dextran in compartments of vesicular shape. After an additional interval of 4 min the number of co-stained structures decreased, while an increase of individually labelled vesicles appeared. Sorting of galectin-3 and dextran was nearly complete later than 10 min of chase with an accumulation of galectin-3 in relatively large endosomal structures, most likely recycling endosomes. In contrast, dextran was segregated into a distinct vesicle population that did not co-localize with galectin-3. The separation between both markers became even more obvious after 20 min of chase. To certify the recruitment of galectin-3 into recycling endosomes, we assessed the co-localization between internalised lectin and Rab11. Here, after 20 min of chase about 35 ± 2% of all galectin-3 fluorescence could be detected in a Rab11positive vesicular recycling compartment (Fig. 7B). Thus suggesting that the fluidphase marker dextran remains in an endosomal compartment, most likely early endosomes as previously described (Leung et al., 2000), while galectin-3 is delivered into subapical recycling endosomes. In addition, we have analyzed the influence of sugar binding on galectin-3 uptake by employing two separate approaches. We used either incubation with lactose, a competitive inhibitor of galectin3–ligand binding, or studied the uptake of the sugar-binding incompetent variant of galectin-3, Gal3R186S . To monitor the collective uptake of galectin-3, fluorescent recombinant galectin-3 or Gal3R186S were added at 4 ◦ C to the apical medium of MDCK cells grown on filters in the absence or presence of carbohydrates. Immediately thereafter surface bound material was fixed for fluorescence microscopy (Fig. 8A and B) or processed for immunoblot analysis (Fig. 8C and D). Internalisation was induced by a 10 min pulse at 37 ◦ C followed by 20 min of chase in the absence of the lectin. As indicated in Fig. 8A and B surface bound galectin-3 could be detected prior to the uptake and was internalised into subapical endosomes after the chase period. However, neither galectin-3 in the presence of 150 mM lactose nor the Gal3R186S mutant bound to the cell surface or accumulated intracellularly by internalisation. This was confirmed biochemically by immunoblot

of the cell lysates before and after internalisation. Here, endogenous canine galectin-3 (30 kDa) could be distinguished from recombinant human galectin-3 (26 kDa) based on the higher molecular weight of the canine homologue (Fig. 8C and D). At first, surface binding of human galectin-3 was not affected by the presence of 300 mM glucose as indicated by the lectin band in lysates of cells before uptake. Following uptake, internalisation resulted in a significant proportion of intracellular human galectin-3 as detected by immunoblot. On the other hand, neither recombinant galectin3 in the presence of 150 mM lactose nor recombinant Gal3R186S were identified in cell lysates before or after internalisation. The quantities of highly expressed endogenous canine galectin-3 as a control were not obviously affected in these uptake experiments. Altogether, these studies suggested that sugar binding of externally added galectin-3 is absolutely required for its attachment to the cell surface. This seems to be a prerequisite for endocytic uptake. The presence of galectin-3 within endosomes of dendritic cells has been previously suggested by Thery et al. Using a proteomic approach they could identify the lectin in exosomes which are related to endosomes (Thery et al., 2001). In epithelial cells endosomal organelles can connect exocytic with endocytic transport events, which has been previously described for basolateral as well as apical pathways of epithelial cells (Ang et al., 2004; Cramm-Behrens et al., 2008; Leitinger et al., 1995; Lock and Stow, 2005). This linkage directly interconnects material derived from the plasma membrane with newly synthesized cargo that has just exited the Golgi apparatus. If recycled and newly synthesized material would then interact in the lumen of endosomal organelles, how can this interaction be regulated? The mannose 6-phosphate receptor has been shown to dissociate from its ligand within the low endosomal pH (Dahms and Hancock, 2002). Our data indicate that galectin-3 also accumulates within acidified compartments and acidification may play a role in galectin-3 dependent apical sorting. This view is supported by data, which show that polarized sorting in epithelial cells depends on an acidic intracellular compartment (Parczyk and Kondor-Koch, 1989; Caplan et al., 1987). The question, how galectin-3 enters this compartment, still remains to be solved. One pathway could involve sugar-dependent endocytic uptake from exogenous galectin-3 already present in the extra-

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Fig. 4. Immunofluorescence analysis of endogenously expressed galectin-3 with Rab4, 8 or 11. MDCKII cells were grown on coverslips for 6 days until cells were fully polarized. After fixation and permeabilization cells were immunostained with anti-galectin-3/Alexa546 and anti-Rab4, 8 or 11/Alexa488 respectively. Recorded epifluorescence image stacks were deconvoluted for background elimination. Images of the subapical area are indicated. Fluorescence staining of galectin-3 and Rab GTPases is shown individually or in merged images. Co-stained structures are indicated by arrows. Scale bars, 10 ␮m.

cellular milieu. Another, not yet identified non-classical secretion mechanism could translocate galectin-3 across endosomal membranes or the plasma membrane (Nickel and Seedorf, 2008). In conclusion, we observed in COS-1 and MDCK cells an enrichment of galectin-3 in Rab-positive endosomes. These include acidified subapical recycling endosomes in MDCK cells, which are also traversed by exocytic apical cargo. Although these findings do not uncover the entire chronology of cellular pathways involved in the exocytosis of galectin-3, insights into participating compart-

ments will aid in the elucidation of non-classical secretion of this lectin. Materials and methods Antibodies Rabbit polyclonal antibodies directed against galectin-3 and mouse monoclonal anti-p75NTR (ME20.4) were kindly provided by

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Fig. 5. TIRFM analysis of galectin-3 at the apical membrane domain of MDCK cells. MDCKIIp75GFP/Gal3DsRed (A and B), MDCKIIp75GFP/Rab11DsRed (C and D), cells and MDCKIIGal3YFP/Rab11DsRed (E and F) cells were grown on filters for 6 days. Filters were excised and positioned upside down on a coverslip for TIRFM. Epifluorescence as well as TIRF images are depicted. In (B), (D), and (F) the penetration depths of the evanescent field was increased in 8 steps from 70 to 200 nm and images were taken for every step. The 8 layers were then processed and used for 3D reconstruction. Blue arrows point to the basal part of the cells, while green and red arrows depict the horizontal plane of the monolayer. Galectin-3 positive fluorescent structures beneath the apical membrane are indicated by arrows. (G) Fully polarized MDCKp75GFP and MDCKGalectin-3DsRed cells were fixed and immunostained with anti-p75/Alexa546 or anti-galectin-3/Alexa488 antibodies before TIRFM analysis. In the upper panel GFP- or in the lower panel DsRed-positive structures that are not immunostained with the corresponding antibody are indicated by arrows. Scale bars, 10 ␮m.

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Fig. 6. Following apical uptake galectin-3 is delivered into acidified organelles. (A) Apical TIRFM analysis of MDCKGal3YFP cells before and after treatment with NH4 Cl. Gal3YFP-positive structures appearing after neutralising endosomal pH are indicated by arrows. (B) Gal3-pHrodo was added to the apical medium of fully polarized MDCK cells at 4 ◦ C. The recombinant protein was internalised at 37 ◦ C and analyzed by fluorescence microscopy for pHrodo fluorescence. A time series from 0 to 45 min of uptake is depicted. The outlines of cells are depicted by grey lines. Scale bars, 10 ␮m in (A), 20 ␮m in (B).

H.P. Elsaesser (University of Marburg, Germany) and Andre LeBivic (IBDM, Marseille). Mouse monoclonal anti-caveolin-1, anti-Rab4, 8 and 11 and rabbit polyclonal anti-GFP antibodies were obtained from BDsciences (Transduction Laboratories, Lexington, KY). Rat monoclonal anti-galectin-3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture, transfection experiments and live cell analysis COS-1 cells were cultured in DMEM (1 g glucose/l), MDCKII cells in DMEM (4.5 g glucose/l) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% FCS at 37 ◦ C in humidified atmosphere containing 5% CO2 . Media for MDCKP75-GFP cells also contained 0.5 mg/ml G418. MDCKIIp75NTRGFP/Gal3DsRed cells were generated by co-transfection with corresponding expression plasmids and selection in MEM medium containing 5% FCS supplemented by G418 (0.5 mg/ml) and Zeocin (0.2 mg/ml). Plasmid transfection of COS-1 or MDCKII cells and subsequent analysis of the cells by immunoblot and confocal microscopy were performed essentially as described before (Delacour et al., 2006; Jacob et al., 2000). Live cell imaging was performed on a Leica DMI6000 B with a HCX PL APO 100× objective. Resulting image stacks were deconvoluted and 3D reconstructed using Volocity (Improvision, Coventry, UK). TGN-exit experiments were performed as previously described (Delacour et al., 2006). For NH4 Cl treatment cells were grown on cover slips with a diameter of 42 mm or on PET filters (BD FalconTM , Franklin Lakes, USA). Excised filters were put upside down into a Bachofer chamber at 37 ◦ C and covered with PBS. After selecting a cell for imaging, PBS was aspirated, borders of the image stack defined and finally the xyzt series started. Immediately after the first time point prewarmed PBS in which NaCl was substituted by an isomolar concentration of NH4 Cl was added. Immunofluorescence and fluorescence microscopy MDCK cells were grown on PET filters (BD FalconTM , Franklin Lakes, USA) in 24 well plates for 6 days. Cells were washed with PBS,

fixed in 4% paraformaldehyde and blocked with 1% BSA. Immunostaining was then performed with primary antibodies as indicated and secondary antibodies conjugated to Alexa488 or Alexa546 (Invitrogen, Carlsbad, USA). Total Internal Reflection Fluorescence microscopy (TIRFM) was performed with a Leica AM TIRF MC setup on a Leica DMI6000 B with a HCX PL APO 100× objective. Analysis of transfected COS-1 cells seeded onto 42 mm cover slips took place in a Bachofer chamber at 37 ◦ C and using PBS to cover the cells. For apical TIRF microscopy cells were grown on PET filters (BD FalconTM , Franklin Lakes, USA) in 24 well plates for 6 days. Filters with fully polarized cells were cut out and put into a Bachofer chamber in upside down orientation. To get the cells into close contact to the coverslip filters were weighed. The system was run by Leica LAS AF software and images were processed by the Volocity software package (Improvision, Coventry, UK). Data evaluation and co-localization analysis was performed with ROI detection of the Leica software in combination with the Volocity (Improvision, Coventry, UK) and Imaris imaging software packages (Bitplane, Switzerland). A co-localization with a Pearson correlation coefficient of more than 0.9 was regarded as significant.

Production of recombinant galectin-3 Recombinant galectin-3, galectin-3R186S , and galectin3R186S /FITC® were kindly provided by H. Leffler (Lund, Sweden). For production of recombinant galectin-3 fused to Alexa Fluor® 633, BL21-Star bacteria containing a galectin-3 expression plasmid were cultured in 200 ml LB medium containing 100 ␮g/␮l Ampicillin at 37 ◦ C overnight. To induce galectin-3 expression 100 ␮l IPTG was added to the medium. After 3 h of incubation at 37 ◦ C cells were harvested and subsequently sonicated 10× for 1 min in MEPBS (PBS containing 4 mM ␤-mercaptoethanol and 2 mM EDTA). To remove cell debris the lysate was centrifuged at 14,000 × g for 30 min at 4 ◦ C. Supernatant was recovered and incubated with 500 ␮l lactosyl-sepharose beads (EY Laboratories, San Mateo, USA) at 4 ◦ C overnight. Following this incubation beads were washed three times in ME-PBS and bound galectin-3 finally eluted using

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Fig. 7. Dextran-Alexa488 and Galectin-3-Alexa633 are cointernalised at the apical cell pole. (A) Dextran-Alexa488 and galectin-3-Alexa633 were internalised for 1 min from the apical medium of polarized MDCK cells. The cells were washed and incubated for the indicated times at 37 ◦ C. Thereafter, surface associated galectin-3 was removed by acetic acid incubation followed by paraformaldehyde fixation. Galectin-3/dextran positive structures were quantified and the proportions relative to total galectin-3 are indicated below. In (B) galectin-3-Alexa633 was internalised into Rab11-GFP transfected cells. Co-localized structures are indicated by arrows. Co-localization between galectin-3 and Rab11 was quantified from three independent experiments with cells fixed immediately before incubation at 37 ◦ C or after 20 min of chase. The measurements indicated below were analyzed using Student’s t-test (***p < 0.001). Scale bars, 10 ␮m.

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Fig. 8. Carbohydrate-dependent endocytosis of galectin-3. 1 ␮M recombinant galectin-3-Alexa633 (A and C) or galectin-3-FITC (WT) and galectin-3R186S -FITC (B and D) were added to the apical medium of polarized MDCK cells grown for 6 days on PET filters followed by incubation on ice for 30 min in the absence (Mock) or presence of carbohydrates (150 mM lactose, 300 mM glucose) followed by endocytosis for the indicated time intervals. When material was endocytosed at 37 ◦ C for 30 min galectin-3 was removed from the cell surface by acetic acid treatment. Acetic acid treatment of the cells before uptake did not result in any significant detection of exogenously added galectin-3 (data not shown). They were either prepared for fluorescence microscopy (A and B) or processed for Western blotting using pAb anti-galectin-3 (C and D). Scale bars represent 20 ␮m. cGal3, endogenous canine galectin-3; hGal3, recombinant human galectin-3.

ME-PBS containing 150 mM lactose. The buffer was changed to MEPBS without lactose by using a PD-10 column (GE Healthcare, Little Chalfont, UK). Conjugation to Alexa Fluor® 633 was performed using the Alexa Fluor® 633 Protein Labeling Kit (Invitrogen, Carlsbad, USA). Conjugation to pHrodo (Invitrogen, Carlsbad, USA) was performed essentially as described in the manual using pHrodo succinimidyl ester. Galectin-3 uptake experiments Fully polarized MDCK cells (day 6) were incubated twice for 30 min in MEM (without FCS) and then washed with cold PBS. 1.5 ␮M recombinant human galectin-3-Alexa633, galectin-3-FITC, galectin-3R186S -FITC, galectin-3-pHrodo, recombinant galectin-3 or 1 mg/ml Dextran-Alexa488 were applied to the apical surface of the cell monolayer. The cells were incubated for 30 min at 4 ◦ C prior to endocytosis at 37 ◦ C. They were rinsed with PBS and incubated at 37 ◦ C for different time intervals. Residual material at the cell surface was removed by 0.2% acetic acid incubation for 2 × 5 min. Cells were then processed for fluorescence microscopy or Western blot.

Acknowledgements We are grateful to W. Ackermann, M. Dienst and Lena Grosse for technical assistance. Petra Haunhorst helped us with the production of recombinant galectin-3. We thank Dr. H.P. Elsaesser (University of Marburg) for supplying us with polyclonal antibody (pAb) anti-galectin-3. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany (grants JA 1033, Graduiertenkolleg 1216 and Sonderforschungsbereich 593). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejcb.2010.07.001. References Ahmad, N., Gabius, H.J., Andre, S., Kaltner, H., Sabesan, S., Roy, R., Liu, B., Macaluso, F., Brewer, C.F., 2004. Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. J. Biol. Chem. 279, 10841–10847. Ang, A.L., Taguchi, T., Francis, S., Folsch, H., Murrells, L.J., Pypaert, M., Warren, G., Mellman, I., 2004. Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells. J. Cell Biol. 167, 531–543.

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