The antibody to GD3 ganglioside, R24, is rapidly endocytosed and recycled to the plasma membrane via the endocytic recycling compartment
Inhibitory effect of brefeldin A and monensin
Ramiro Iglesias-Bartolome´ , Pilar M. Crespo, Guillermo A. Gomez and Jose L. Daniotti
Centro de Investigaciones en Quı´mica Biolo´ gica de Co´ rdoba, CIQUIBIC (UNC-CONICET), Departamento de Quı´mica Biolo´ gica, Universidad Nacional de Co´ rdoba, Argentina
Keywords endocytic recycling; gangliosides; glycolipid antibodies; intracellular transport; R24 antibody
Correspondence J.L. Daniotti, CIQUIBIC (UNC-CONICET), Departamento de Quı´mica Biolo´ gica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, Ciudad Universitaria, 5000 Co´ rdoba, Argentina Fax: +54 3514334074 Tel: +54 3514334171 E-mail: daniotti@dqb.fcq.unc.edu.ar
(Received 15 January 2006, revised 14 February 2006, accepted 20 February 2006)
doi:10.1111/j.1742-4658.2006.05194.x
Gangliosides are sialic acid-containing glycosphingolipids present on mam- malian plasma membranes, where they participate in cell-surface events such as modulation of growth factor receptors and cell-to-cell and cell-to- matrix interactions. Antibodies to gangliosides have been associated with a wide range of clinically identifiable acute and chronic neuropathy syn- dromes. In addition, antibodies to tumor-associated gangliosides are being used as therapeutic agents. Their binding to and release from cell mem- branes and intracellular destinations have not so far been extensively exam- ined. In this study, we characterized in both GD3 ganglioside-expressing Chinese hamster ovary (CHO)-K1 and SK-Mel 28 melanoma cells the intracellular trafficking and subcellular localization of the mouse monoclo- nal antibody to GD3, R24. By biochemical techniques and detailed confo- cal microscopic analysis, we demonstrate that the GD3–R24 antibody complex is rapidly and specifically internalized by a dynamin 2-independent pathway and then accumulates in the endocytic recycling compartment. In addition, we show that the R24 antibody exits the recycling compartment en route to the plasma membrane by a dynamin 2-dependent pathway sen- sitive to brefeldin A and monensin. Taken together, our results indicate that the GD3–R24 complex is endocytosed in GD3-expressing cells, accu- mulates in the recycling endosome, and is transported back to the plasma membrane via a route that involves clathrin-coated vesicles.
[6]. After
Abbreviations BFA, Brefeldin A; CHO, Chinese hamster ovary; DMEM, Dulbecco’s modified Eagle’s medium; GFP, green fluorescent protein; GalNAc-T, UDP-GalNAc–LacCer ⁄ G3 ⁄ GD3 N-acetylgalactosaminyltransferase; GD3, NeuAca2,8NeuAca2,3Galb1,4Glc ceramide; GM3, NeuAca2,3Galb1,4Glc ceramide; Sial-T2, CMP-NeuAc–GM3 sialyltransferase; TGN, trans-Golgi network; YFP, yellow fluorescent protein.
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Gangliosides are complex glycosphingolipids contain- ing one or more sialic acid residues, which are mainly located at the outer leaflet of the plasma membrane of eukaryotic cells. They participate in cell-surface events such as modulation of growth factor receptors and cell-to-cell and cell-to-matrix interactions [1–5]. They are synthesized in the lumen of the Golgi complex by a complex system of membrane-bound glycolipid acceptors, glycosyltransferases, and sugar nucleotide synthesis, gangliosides their transporters leave the Golgi complex via the lumenal surface of transport vesicles. We recently demonstrated that the ganglioside NeuAca2,8NeuAca2,3Galb1,4Glc ceramide (GD3) is transported from the trans-Golgi network to the plasma membrane via a Rab11- (TGN) exocytic independent, brefeldin A (BFA)-insensitive
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Results
GD3–R24 antibody complex is rapidly internalized in GD3-expressing CHO-K1 cells
pathway [7]. After their arrival at the plasma mem- brane, gangliosides undergo endocytosis. Once inter- nalized, glycosphingolipids can be: (a) recycled to the plasma membrane directly from early endosomes; (b) sorted from endosomes to the Golgi complex, where they can be reglycosylated; or (c) degraded at the lyso- somal level [8]. clone
In addition, antibodies
Antibodies to more than 20 different glycolipids, including gangliosides, have been associated with a wide range of clinically identifiable acute and chronic neuropathy syndromes, including Guillain–Barre´ syn- drome (anti-GM1, anti-GM2, anti-GQ1b), chronic idiopathic ataxic neuropathy (anti-GM3, anti-GD1b, anti-GD3, anti-GQ1), Miller–Fisher syndrome (anti- GQ1b) and multifocal motor neuropathy (anti-GM1) [9,10]. to tumor-associated gangliosides are being used as therapeutic agents, for example, anti-GD2 for neuroblastoma [11] and anti- GD3 for melanoma [12,13]. Several targeted therapies need the antibody to remain at the cell surface to mediate antibody-dependent and complement-depend- ent cytotoxicity. However, rapid internalization of the antibodies to intracellular compartments is desired for cytotoxic effects of toxins or cytotoxic drugs conju- gated with the antibody [14]. Neither the binding and release of ganglioside antibodies from cell membranes nor their intracellular destinations have so far not been extensively tested. In this
in cells
that demonstrate analysis, we
We established an antibody-binding technique to track the fate of surface GD3–R24 after its internalization in a GD3-expressing CHO-K1 cell (clone 2), already established in our laboratory by stable expres- sion of CMP-NeuAc–GM3 sialyltransferase (Sial-T2) cDNA [7,15,16]. Briefly, cells from clone 2 were incu- bated for 10 min on ice to inhibit intracellular trans- port and then with R24 [17] for 45 min on ice. Then, cells where extensively washed with cold buffer to remove unbound antibody, and the temperature chan- ged to 37 (cid:1)C to restore transport and thereby allow endocytosis of GD3–R24 for different times. Confocal microscopic analysis revealed that R24 bound to live cells at 4 (cid:1)C had a plasma membrane punctate distri- bution (Fig. 1A), as previously demonstrated in this cell line [18]. After induction of endocytosis by chan- ging the temperature to 37 (cid:1)C, GD3–R24 was found in vesicles all over the cytoplasm, and at 15 min it began to show a perinuclear distribution. After 30 min at 37 (cid:1)C, the intracellular pool of R24 became more con- centrated in the perinuclear region and the plasma membrane mark almost disappeared (Fig. 1A). At 60 min after the beginning of endocytosis, almost all cells were negative for R24 staining unless the anti- body was present constantly in the cell culture med- ium. It should be mentioned that, at the steady-state, most of the GD3 ganglioside was found to be present in the plasma membrane from clone 2, although a fraction was also observed in endosomal structures [18]. Endocytosis of R24 seems to be specif- ically mediated by GD3, as wild-type CHO-K1 cells, which only express the GM3 ganglioside, did not bind (Fig. 1B, 45 min at 4 (cid:1)C) and internalize R24, even with antibody in the culture medium for 2 h at 37 (cid:1)C (Fig. 1B).
the antibody was
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Next, we performed a western blot to quantify the proportion of endocytosed R24 at different times (Fig. 1C). Thus, cells were incubated with R24 at 4 (cid:1)C to label the surface pool of GD3 ganglioside, and then shifted to 37 (cid:1)C to restore transport. Cells at 15, 30, 60 and 90 min were acid stripped to remove mem- brane-bound antibody, harvested, and the presence of internalized R24 analyzed by western blot. To evaluate the efficiency of surface stripping by acid washing, one sample was acid-treated directly after incubation at 4 (cid:1)C; under these conditions the antibody was com- pletely removed from the cell surface (result not shown). About 50% of the antibody bound to the cell study, we characterized in both GD3- expressing Chinese hamster ovary (CHO)-K1 and SK- Mel 28 melanoma cell lines the binding, intracellular trafficking, and subcellular localization of the mouse monoclonal antibody to GD3, R24(IgG3). By bio- chemical techniques, immunofluorescence and confocal microscopic the GD3–R24 complex was rapidly and specifically inter- nalized and accumulated mainly in a perinuclear compartment. This compartment was positive for expression of the GTPase Rab11 and internalized transferrin, which are two recycling endosome mark- ers, but not for UDP-GalNAc–LacCer ⁄ G3 ⁄ GD3 N-acetylgalactosaminyltransferase (GalNAc-T), a TGN marker. In addition, we show that R24 exited the recycling compartment by a dynamin 2-dependent pathway sensitive to BFA and monensin. Interestingly, after 60 min of endocytosis of the R24 antibody, most recovered from the culture of medium. Taken together, our results indicate that the GD3–R24 complex is rapidly endocytosed in CHO-K1 and SK-Mel 28 melanoma cells, accumulates in the recycling endosome, and is transported back to the plasma membrane via a route involving clathrin-coated vesicles.
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A
B
C
Fig. 1. R24 is rapidly and specifically endocytosed in GD3-expressing CHO-K1 cells. (A) CHO-K1 cells (GD3+) were incubated with R24 for 45 min at 4 (cid:1)C. After washing of the cells, the temperature was shifted to 37 (cid:1)C to allow endocytosis of the complex GD3–R24, and cells were fixed at 15, 30 and 60 min. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa488. Single confocal sections of 0.7 lm were taken parallel to the coverslip. Cell boundaries (white lines) are indicated at 30 and 60 min. The perinuclear region is also indic- ated (arrows) at 15 and 30 min. The fluorescence micrographs shown are representative of five independent experiments. (B) Wild-type CHO-K1 cells (wt, GD3–) were incubated with R24 for 45 min at 4 (cid:1)C. Then the temperature was shifted to 37 (cid:1)C, and cells were fixed at 2 h. R24 antibody detection was carried out as indicated in (A). (C) Cells from clone 2 (GD3+) were incubated with R24 at 4 (cid:1)C to label the surface pool of GD3 ganglioside, and then shifted to 37 (cid:1)C to restore transport. Cells at 15, 30, 60 and 90 min were acid stripped to remove membrane-bound antibody, harvested, and the presence of internalized R24 analyzed by western blot. The expression of Sial-T2–hemagglut- inin (GD3 synthetase) in the same membrane was analyzed as a control of protein loading. IgG (L), Light chain of IgG. Scale bars: 20 lm.
that
also obtained at 15 and 45 min after endocytosis (results not shown). R24 was found colocalized to a minor extent with the acidotropic probe in small acidic organelles, which probably represents endo- little R24 anti- somes. These results suggest body enters lysosomes or that the epitope for the R24 antibody is rapidly lost on transport into lyso- somes.
surface at 4 (cid:1)C was efficiently endocytosed after 15 min at 37 (cid:1)C. After 30 min, the pool of intracellular antibody had decreased to 25%, and, at 60 and 90 min after internalization, the antibody concentration was below the limit of detection by western blot. These results confirm the immunofluorescence and confocal microscopic findings. Taken together, the results show that R24 is rapidly internalized in GD3-expressing CHO-K1 cells, intracellularly accumulated, and later degraded and ⁄ or depleted from subcellular compart- ments.
R24 antibody is not targeted and degraded in lysosomes
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To evaluate further whether the antibody is degra- ded in lysosomes, internalization and intracellular transport of R24 was performed in the presence of NH4Cl and chloroquine, which are inhibitors of lyso- some degradation. Cells from clone 2 were allowed to internalize R24 in the absence (control) or presence of 30 mm NH4Cl or 60 lm chloroquine over 90 min at 37 (cid:1)C. Cells were then harvested and the presence of R24 analyzed by western blot. As clearly shown in the inhibitors could not prevent cellular Fig. 2B, depletion of R24 antibody. Taken together, these the R24 antibody is results indicate that most of not targeted to and degraded in lysosomes after its internalization. To assess whether the R24 antibody was targeted to lysosomes during endocytic transport, we performed a colocalization analysis of internalized antibody with the acidotropic probe LysoTracker Red. Results shown in Fig. 2A clearly indicate that endocytosed R24 did not significantly colocalize with the lyso- some marker at 30 min. Comparable results were
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A
B
Fig. 2. R24 antibody is not degraded in lysosomes. (A) CHO-K1 cells (GD3+) were incubated with R24 for 45 min at 4 (cid:1)C. After washing of the cells, the temperature was shifted to 37 (cid:1)C to allow endocytosis of the complex GD3–R24, and cells were fixed at 30 min. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa488. For lysosome staining, cells were incubated with 0.2 lM acidotropic probe LysoTracker Red DND-99 for 15 min at 37 (cid:1)C before fixation. Single confocal sections of 0.7 lm were taken parallel to the coverslip. Cell boundaries (white lines) are indicated. Scale bar: 20 lm (B) GD3-expressing CHO-K1 cells were incubated with R24 for 45 min at 4 (cid:1)C (0 min). After washing of the cells, the temperature was shifted to 37 (cid:1)C to allow internalization of R24 antibody in the absence or presence of 30 mM NH4Cl or 60 lM chloroquine over 90 min at 37 (cid:1)C. Then cells were harvested and the presence of R24 antibody analyzed by west- ern blot. The expression of Sial-T2–hemagglutinin (GD3 synthetase) in the same membrane was analyzed as a control of protein loading.
The internalized R24 antibody is targeted and transiently accumulated at the recycling endosome
that does not overlap with transferrin and Rab11 would be associated with different recycling endosome membranes, as it has been suggested, on the basis of cellubrevin and endocytosed transferrin juxtanuclear localization, that these compartments may be subdivi- ded into distinct populations [20].
The R24 antibody is recycled to the plasma membrane and released into the culture medium
to the plasma membrane where it As shown previously, we found that most of the endo- cytosed R24 was not targeted to lysosomes but transi- ently accumulated at the recycling endosome. After 60 or 90 min of R24 endocytosis, we could not detect the internalized antibody by biochemical and immunologi- cal techniques. An explanation for these results is that R24 may be recycling from the pericentriolar endocytic compartment is released into the culture medium.
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After internalization, a significant fraction of R24 was located in a perinuclear region that resembles the Golgi complex or the recycling endosomes as these or- ganelles have a pericentriolar distribution in CHO-K1 cells [19]. In an effort to identify the juxtanuclear com- partment where the endocytosed R24 antibody is con- centrated, we performed extensive colocalization with markers of both recycling endosome and Golgi com- plex (Fig. 3). After 30 min of internalization, no colo- calization was observed between R24 and GalNAc-T fused to yellow fluorescent protein (YFP), a TGN marker. However, we observed extensive colocalization between R24 and the GTPase Rab11, an established recycling endosome marker [19]. In addition, we also found substantial overlapping of R24 with coendocyto- sed Alexa647-transferrin in a perinuclear compartment, demonstrating that a significant fraction of endocyto- sed R24 in CHO-K1 cells was present in the recycling endosome. The fraction of perinuclear labeling of R24 To address this issue, cells from clone 2 were incuba- ted for 10 min on ice to inhibit intracellular transport and then with R24 for 45 min on ice. Afterwards, cells were allowed to internalize the antibody for 20 min at
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Fig. 3. The internally accumulated R24 antibody colocalizes with recycling endosome markers but not with the Golgi marker GalNAc-T. CHO-K1 cells (GD3+) transiently expressing both GalNAc-T-YFP (upper row, pseudo colored green) and wild-type GFP-Rab11 (GFP-Rab11 wt, middle row) were incubated with anti-GD3 IgG for 45 min at 4 (cid:1)C. After washing of the cells, the temperature was shifted to 37 (cid:1)C to allow endocytosis of the complex GD3–R24 for 30 min. R24 antibody was detected by using rhodamine-conjugated goat anti-mouse IgG. In another set of experiments, uptake of Alexa647-transferrin (Alexa647-Tf, pseudo colored green) was monitored simultaneously with R24 endocytosis (lower row). Expression of Rab11 and GalNAc-T was detected by the intrinsic fluorescence of GFP and YFP, respectively. Cell boundary (white line) is indicated in the upper row. Insets in merge panels show details at higher magnification. The fluorescence micro- graphs shown are representative of three independent experiments. Scale bars: 10 lm.
60 min most of it was recovered from the culture med- ium. The antibody recovered from the culture medium was found to have the expected molecular mass (whole molecule) in gels run under nonreducing conditions (results not shown). Together, these results indicate that R24, once internalized, is recycled to the plasma membrane and released into the culture medium.
R24 antibody recycling is sensitive to BFA and dependent on dynamin activity
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It has previously been shown that transferrin receptor recycling as well as the formation of clathrin-coated 37 (cid:1)C, and then the temperature was changed again to 4 (cid:1)C. The cell surface was then stripped of any remain- ing antibody with acid wash. At this point, cells only contained R24 in intracellular compartments. Subse- quently, prewarmed culture medium was added to the cells, which were maintained at 37 (cid:1)C to restore intra- cellular transport. Cells and culture medium were recovered at different times, and the presence of R24 in both samples was analyzed by western blot. As shown in Fig. 4, at the beginning of the time-course experi- ment (stripped cells, 0 min) the antibody was present only in the cell fraction. At 15 min, it was detected in both fractions (cells and culture medium), and at
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A
experiments
B
Fig. 4. R24 antibody is recycled to the plasma membrane and released into the culture medium. (A) GD3-expressing CHO-K1 cells were incubated with R24 antibody for 45 min on ice. Afterwards, cells were allowed to internalize the antibody for 20 min at 37 (cid:1)C, and then the temperature was shifted again to 4 (cid:1)C. The cell sur- face was then stripped of any remaining antibody with acid wash (0 min). Cells were then incubated at 37 (cid:1)C to restore intracellular transport, and cells and culture medium were recovered at 15 and 60 min. The presence of R24 antibody in both samples was ana- lyzed by western blot as indicated in Experimental procedures. (B) The relative contribution of bands in each condition was calculated using the computer software SCION IMAGE on the scanned film shown in (A). Ponceau S staining was used to normalize levels of proteins seeded in each lane. The band intensity for R24 antibody at 0 min (cellular fraction) was arbitrarily taken as 1. Results are representative of four independent experiments.
however, in BFA-treated cells a significant fraction of the antibody remained accumulated in a perinuclear compartment, consistent with the requirement of clath- rin-coated vesicles for efficient R24 recycling. The intracellular accumulation of R24 at 90 min was also detectable by western blot (Fig. 5B). Under these conditions, we also found in BFA-treated cells substantial overlapping of R24 with co-endocyto- sed Alexa647-transferrin and with GalNAc-T-YFP, a TGN marker (Fig. 5C, first row). These results clearly demonstrate a fusion between the recycling endosomal system and the TGN in the presence of BFA, as previ- ously described [24].
Dynamins function in the pinching off of clathrin- coated vesicles. It has previously been demonstrated that the transferrin receptor egresses recycling endo- somes, at least in part, by endosome-derived clathrin- coated vesicles in a dynamin-dependent manner [22]. To study the requirement for dynamin in R24 depar- ture from recycling endosome, GD3-expressing CHO- K1 cells were transiently transfected to express both wild-type (wtDyn2) and the dominant-negative form of dynamin-2 (Dyn2K44 A), a mutant defective in GTP loading and hydrolysis. After 24 h, cells were incuba- ted with R24 at 4 (cid:1)C for 45 min and then induced to internalize the antibody by shifting the temperature to 37 (cid:1)C. Cells were fixed at 30, 60 and 90 min, and the presence of R24 was visualized by immunofluorescence and confocal microscopic analysis. Results shown in Fig. 6 demonstrate that wild-type dynamin-2 did not affect R24 internalization (30 min) and later depletion (60 and 90 min). On the other hand, the dominant- negative version of dynamin-2 did not have much effect on R24 internalization (30 min), but it signifi- cantly affected the departure of R24 from the recycling endosome (60 and 90 min). Taken together our data indicate that recycling endosome-derived, clathrin-coa- ted vesicles play a role in the endosomal recycling pathway of the R24 antibody.
R24 antibody recycling is also sensitive to monensin
structures that move pits on the recycling endosome is inhibited in the pres- ence of BFA [21–23]. If clathrin-coated vesicles play a role in R24 recycling, BFA should interfere with this this hypothesis, GD3-expressing pathway. To test CHO-K1 cells were incubated on ice with R24 for 45 min. The cells were then incubated on ice in the absence (control) and presence of 2 lgÆmL)1 BFA for 15 min. At the end of this period, cells where washed to remove unbound antibody, and then prewarmed culture medium supplemented with BFA, when neces- sary, was added, and the cells were transferred to 37 (cid:1)C to allow endocytosis for different times.
endosomal
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It was previously proposed that endosomal acidifica- tion is a prerequisite for the actual formation of the carrier the TGN proteins (TGN38 or furin) from the endosome back to the TGN [25]. In an attempt to learn more about the mechanism of recycling of R24, we examined the effect of monensin, an ionophore that dissipates pH gradients across organelle membranes [26], on R24 recycling. Results shown in Fig. 5A indicate that BFA did not affect R24 internalization, because the fraction of internalized and accumulated antibody at 30 min was similar in both control and BFA-treated cells. As internalization we shown previously, 90 min after could not detect the presence of intracellular antibody;
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trol) or presence of 10 lm monensin. At the end of this period, the unbound antibody was removed by washing, and internalization was allowed to continue GD3-expressing CHO-K1 cells were incubated with the antibody for 45 min on ice. Then cells were incu- bated on ice for another 15 min in the absence (con-
A
B
C
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Fig. 6. R24 antibody recycling is dependent on dynamin activity. GD3-expressing CHO- K1 cells were transiently transfected to express both wild-type (wtDyn2) and the dominant-negative form of dynamin-2 (Dyn2K44 A). After 24 h, cells were incub- ated with the R24 antibody at 4 (cid:1)C for 45 min and then allowed to internalize the antibody by shifting the temperature at 37 (cid:1)C. Cells were fixed at 30, 60 and 90 min, and the presence of R24 analyzed by using rhodamine-conjugated goat anti- mouse IgG. Single confocal sections of 0.7 lm were taken parallel to the coverslip. Arrows indicate dynamin-transfected cells. Cell boundaries (white lines) are indicated at 30, 60 and 90 min. Scale bars: 10 lm.
Fig. 5. R24 antibody recycling is sensitive to BFA and monensin. (A) GD3-expressing CHO-K1 cells were incubated on ice with the R24 anti- body for 45 min. Then cells were incubated on ice in the absence (control) and presence of 2 lgÆmL)1 BFA or 10 lM monensin for 15 min. Cells where washed to remove unbound antibody and then prewarmed culture medium supplemented with BFA or monensin, when neces- sary, was added and cells transferred to 37 (cid:1)C to allow endocytosis for 30 and 90 min. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa488. Single confocal sections of 0.7 lm were taken parallel to the coverslip. Cell boundaries (white lines) are indic- ated at 30 and 90 min. (B) GD3-expressing CHO-K1 cells were incubated with R24 for 45 min at 4 (cid:1)C (0 min). After washing of the cells, the temperature was shifted to 37 (cid:1)C to allow internalization of R24 antibody in the absence (C) or presence of 2 lgÆmL)1 BFA or 10 lM monen- sin (Mon) over 30 and 90 min at 37 (cid:1)C. Then cells were harvested and the presence of R24 antibody was analyzed by western blot in sam- ples containing equal amounts of total proteins. (C) Same experiment as in (A) except that GalNAc-T-YFP was transiently expressed 24 h before R24 and Alexa647-transferrin (Alexa647-Tf) internalization. Insets in merge panels show details at higher magnification. Triple color imag- ing of single fixed CHO-K1 cells were taken with filters for rhodamine, Cy5 and YFP (first, second, and third panels from left, respectively). The fourth panel is a merging of these images. Triple colocalization is visualized in white, and colocalization between R24 antibody and Alexa647-transferrin is visualized in pink. Scale bars: 10 lm.
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at 37 (cid:1)C for different times. Results shown in Fig. 5A indicate that monensin did not affect R24 internalizat- ion, as it was similar in both control and treated cells at 30 min. As described above, at 90 min after inter- nalization we could not detect intracellular antibody in untreated cells; however, in monensin-treated cells a significant fraction of the antibody remained accumu- lated in a perinuclear compartment. The intracellular
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accumulation of R24 in monensin-treated cells was also observed in western blot experiments at 30 and 90 min (Fig. 5B). We also found in monensin-treated cells overlapping of R24 antibody with co-endocytosed Alexa647-transferrin. However, in contrast with the effect of BFA, in monensin-treated cells we only observed a slight colocalization between R24 antibody and GalNAc-T-YFP, a TGN marker (Fig. 5C, second row). Our initial interpretation of these results is that they indicate that, like TGN38 and the TGN protease furin, endosomal acidification is probably required for R24 antibody to exit the recycling endosome.
R24 antibody is also internalized, recycled to plasma membrane, and released into culture medium in SK-Mel 28 melanoma cells sides expressed in this cell line, which run as doublets because of differences in ceramide structure. Melan- oma cells were incubated with R24, the unbound anti- body was removed by washing, and internalization was induced by transferring the cells to 37 (cid:1)C for dif- ferent periods of time. As shown in Fig. 7B, R24 was efficiently internalized in SK-Mel 28 cells at 15, 30 and 60 min. Colocalization between endocytosed R24 and transferrin was observed after 30 min (results not shown). In this cell line, tubules from endocytic recyc- ling are distributed more widely throughout the cyto- plasm. Compared with in CHO-K1 cells, the antibody showed a lower rate of intracellular disappearance. In western blot experiments we observed that, at 60 and 90 min, a fraction of the antibody still remained asso- ciated with intracellular structures whereas a significant fraction was found in the culture medium (Fig. 7C).
Discussion
labeling pattern of gangliosides
We have followed the entire endocytic itinerary of the mouse monoclonal antibody to GD3, R24 in GD3-expressing CHO-K1 and SK-Mel 28 cells. We found that endocytosed R24 first appears in a diffuse, To investigate if the internalization and recycling path- way of R24 antibody is a common feature that occurs in other cell types, we analyzed the endocytic transport of this antibody in SK-Mel 28 melanoma cells. The from radioactive SK-Mel 28 cells is shown in Fig. 7A. As previously reported [17], GD3 and GM3 were the major ganglio-
B
A
C
Fig. 7. R24 antibody is internalized, recycled to the plasma membrane, and released into culture medium in SK-Mel 28 melanoma cells. (A) SK-Mel 28 melanoma cells were metabolically labeled from [14C]galactose for 24 h. Lipid extracts were prepared, purified, chromatographed and visualized as indicated in Experimental procedures. The positions of cochromatographed glycolipid standards are indicated. The asterisk indicates the position of an unidentified lipid. (B) SK-Mel 28 melanoma cells were incubated with R24 for 45 min at 4 (cid:1)C. After washing of the cells, the temperature was shifted to 37 (cid:1)C to allow endocytosis of the complex GD3–R24, and cells were fixed at 15, 30 and 60 min. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa488. Single confocal sections of 0.7 lm were taken parallel to the coverslip. (C) SK-Mel 28 melanoma cells were incubated with R24 antibody for 45 min on ice. Cells were allowed to internalize the antibody for 20 min at 37 (cid:1)C, and then the temperature was shifted again to 4 (cid:1)C. The cell surface was then stripped of any remaining antibody with acid wash (0 min). Then cells were incubated at 37 (cid:1)C to restore intracellular transport, and cells and culture medium were recovered at 30, 60 and 90 min. The presence of R24 antibody in both samples was analyzed by western blot as indicated in Experimental procedures. Scale bars: 20 lm.
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punctate distribution in the cytoplasm that is consis- tent with the sorting compartment of the early endo- somes. Subsequently, R24 appeared concentrated in a pericentriolar distribution which we have characterized as the recycling endosome. After that, the antibody is recycled to the plasma membrane and released into the culture medium by a BFA ⁄ monensin-sensitive, dynam- in-dependent recycling pathway. In addition, no evi- dence was found for targeting and degradation of R24 antibody in lysosomes. These observations suggest that R24 follows an endocytic pathway typical of other recycling proteins, such as the model recycling protein transferrin receptor. Nevertheless, in this study we des- cribe for the first time the entire endocytic itinerary of a glycolipid antibody.
the
endogenous
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Antibody-binding techniques are extensively used to follow endocytic transport of proteins such as glycosylhoshatidylinositol-anchored proteins [27–29], major histocompatibility complex class I protein and interleukin 2 a-subunit receptor [30], integrin b1 [31] and cation-independent mannose 6-phosphate receptor [32]. Antibodies tend not to have a significant effect on the endocytic behavior of the proteins studied [31,32]. Lysosome and endosome compartments have an acidic pH, which both promotes the dissociation of ligands such as low-density lipoprotein from their receptors and the proper function of hydrolytic enzymes. We demonstrate that the association of R24 with the GD3 ganglioside was unaffected even after 1 h at pH 5 or 6 (Fig. S1), suggesting that the GD3– R24 complex could not be disrupted in acidic organ- elles. Taking all these antecedents together, it is plaus- ible that the itinerary of the R24 antibody reflects the intracellular transit of the disialo ganglioside GD3. In this vein, it has been demonstrated that exogenous glycosphingolipids can be internalized and directed to the Golgi apparatus, where they can be reglycosylated and then delivered back to the cell surface [8,33]. Also, internalized sphingolipid analogs (BODIPY-and NBD- labeled lipids) can be recycled to the plasma mem- brane via endosomes or through the Golgi complex [34,35]. However, it should be taken into consideration that the quantitative and qualitative behavior of the analog lipids is quite different from long-chain cellular lipids, as the ability of short fluorescent lipids to diffuse spontaneously between different membranes is lipid not generally shared by their counterparts [36]. Even the binding of antibodies and toxins to endogenously synthesized lipids may perturb the natural behavior of these molecules. In a similar it was reported that cholera toxin alters the way, internalization mechanism of a fluorescent GM1 ganglioside [37]. It has been reported that transferrin receptor and its ligand transferrin recycle to the plasma membrane with the same kinetics as certain lipids [34] and independ- ently of the transferrin receptor cytoplasmic domain [38]. These data suggest that recycling of molecules from endosome to plasma membrane can occur without active recruitment of cytosolic coat proteins (bulk flow process). However, more recently dynamin-dependent recycling by endosome-derived transferrin receptor clathrin-coated vesicles was reported [22]. Supporting this observation, results obtained in this work using BFA and the dominant-negative form of dynamin recycling endosome-derived, clathrin- indicate that coated vesicles may play a role in the endosomal recyc- ling pathway of the R24 antibody. Previous studies using a cross-linkable form of clathrin light chain indi- cated that, once internalized, the return of the transfer- rin receptor to the cell surface was largely insensitive to clathrin cross-linking [39], consistent with the notion that clathrin does not play a role in trafficking mole- cules from the endosome back to the plasma mem- brane. However, these results do not entirely exclude the possibility that clathrin-coated pits may be oper- ating in the transport of transferrin receptor back to the plasma membrane in the absence of the cross- recycling linker. Alternatively, clathrin independent pathways may also be involved in the recycling of molecules to the plasma membrane, as discussed below. If it is assumed that R24 is associated with the luminal membrane-bounded GD3 during the recycling pathway, segregated into complex must be specialized domains to be sequestered by nascent clath- rin-coated vesicles. Three properties are key to lipid sorting: headgroup interactions, lipid shape and mem- brane-order parameters [40]. GD3–R24 interactions with a protein may cause the lipid to be sorted on the basis of the characteristics of the protein, and such a for the trafficking of gly- mechanism is important cosylphosphatidylinositol-anchored proteins [41]. We recently demonstrated that GD3 ganglioside, like gly- cosylphosphatidylinositol-anchored proteins, is mainly expressed in glycosphingolipid-enriched microdomain (also called detergent-resistant membranes or rafts), dynamic assemblies of cholesterol, saturated phospho- lipids, and sphingolipids [16,18]. The partition of GD3 into glycosphingolipid-enriched microdomains may represent a positive sorting signal for the correct sub- cellular trafficking, as previously described for other lipid-anchored proteins [42,43]. Finally, it is known that dynamin participates in both clathrin-mediated endocytosis and caveolae internalization [37]. Thus, the lack of effect of the dominant-negative form of dynamin on R24 internalization (Fig. 6) suggests that
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Endocytic trafficking of an antibody to GD3
the GD3–R24 complex may occur endocytosis of through clathrin-independent and caveolar-independ- ent processes.
into the extracellular medium through vesicle-mediated secretion (exosomes) as R24 present in the culture medium was not pelleted by centrifugation at 100 000 g (Fig. S3). Further work is required to define the precise mechanism involved in the release of R24 antibody into the extracellular medium.
The recycle pathway described in this work is likely to be of considerable biological and immunological significance. Antibodies to GD3 are being used as thera- peutic agents for melanoma [13]. However, the rapid internalization of R24 antibody observed in GD3- expressing cells may be potentially detrimental to its therapeutic use as it could not be linked to pathways of complement-dependent and cellular-dependent anti- cancer activity. It would be possible to exploit the internalization feature for selective delivery of cyto- toxic agents to GD3-expressing cancer cells. Finally, the results of this study may provide a better under- standing of how antibodies to glycosphingolipids associated with neuropathies harm the different com- partments of the peripheral nerve [9]. However, more information is needed about the rate and routes of intracellular transport of antibodies to glycosphingo- lipids associated with neurological disorders.
Experimental procedures
Cell lines, cell culture and DNA transfection suggesting strongly
liposomes
The following cells were used: wild-type CHO-K1 cells (ATCC, Manassas, VA, USA); CHO-K1 clone 2, a stable Sial-T2 (tagged at the C-terminus with the nanopeptide epi- tope of the viral hemagglutinin) transfectant expressing the ganglioside GD3 [15,16]; and the SK-Mel 28 melanoma cell line (ATCC). Cells were grown and maintained at 37 (cid:1)C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. transfections were carried out with 1 lg per Transient 35-mm dish using cationic (Lipofectamine; Invitrogen, Carlbad, CA, USA) essentially according to the manufacturer’s instructions and incubated for 24 h at 37 (cid:1)C with the transfection reagent and DNA mixture.
Results from this work indicate that BFA-treated cells failed to recycle a fraction of endocytosed R24 antibody. A simple interpretation of these results is that R24 antibody may recycle via a single route and that BFA is inefficient or reduces the rate of R24 recycling. However, another explanation is that two recycling pathways might be operating for R24 anti- body, as previously demonstrated for endocytosed transferrin receptor [23]. The first involves passage through a recycling endosome. Transport from this compartment to the plasma membrane involves clath- rin-coated vesicles which bud off from the recycling endosome. Results indicate that BFA and the domin- ant-negative form of dynamin interfere with this path- way. The second recycling pathway bypasses the recycling endosome and involves direct transfer from the sorting endosome to the plasma membrane. On this subject, we demonstrate that R24 antibody is endocytosed and accumulated in sorting endosomes at 16 (cid:1)C colocalizing to some extent with the GTPase Rab5 (Fig. S2). A similar analysis is valid for the inter- pretation of results obtained using the carboxylic iono- phore monensin because, in monensin-treated cells, a proportion of endocytosed R24 antibody failed to recycle to the plasma membrane. This intracellular pool of R24 was found overlapping with co-endocyto- sed Alexa647-transferrin, that monensin perturbs the departure of R24 from the recycling endosome and hence that endosomal acidifi- cation is probably required for the formation of the carrier structures that move R24.
The GTPase Rab11a–GFP wild-type construct was kindly provided by M. Colombo (Universidad Nacional de Cuyo, Mendoza, Argentina); plasmids coding for wild-type dyn- amin-2 (wtDyn2), dynamin-2 K44A (Dyn2K44 A) and GFP-Rab5 were supplied by J. Bonifacino (NICHD, National Institutes of Health, Bethesda, MD, USA). The construct containing the cDNA coding for the N-terminal domain (cytosolic tail, transmembrane domain, and a few
Expression plasmids
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Results from this work also indicate that R24, once internalized, is recycled to the plasma membrane and released into the culture medium. How can it be explained that R24 is not associated with the plasma membrane after recycling and release into the extracel- lular medium? If R24 antibody is associated with GD3 during the entire endocytic trafficking, the complex could be dissociated in the plasma membrane probably by a shift in the equilibrium in the absence of a signifi- cant concentration of free antibody in the medium. However, this appears improbable, as we showed that the association of R24 with GD3 was unaffected over 1 h at pH 7.4 (Fig. S1). On the other hand, evidence is accumulating that supports a physiological role for exosomes in the removal of the transferrin receptor during reticulocyte development, although exosomes have been isolated from the cell culture of many non- haematopoietic cells [44]. However, we also exclude the possibility that the GD3–R24 complex is released
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The presence of R24 in cells and culture medium was assessed by western blot.
amino acids from the stem region) of GalNAc-T fused to the N-terminus of the YFP (GalNAc-T-YFP) was obtained by subcloning the corresponding cDNA fragments into the plasmid pEYFP-N1 (Clontech, Mountain View, CA, USA) [6].
R24 recycling in BFA-treated and monensin-treated cells
Clone 2 cells were incubated on ice for 10 min to inhibit intracellular transport and then with R24 antibody for 60 min with 2 lgÆmL)1 BFA or 10 lm monensin present in the culture medium for the last 15 min. Subsequently, cells were washed three times with cold NaCl ⁄ Pi and transferred to 37 (cid:1)C with fresh prewarmed complete DMEM supple- mented or not with BFA or monensin as indicated. At dif- ferent times, cells were harvested or fixed as described below. The presence of the R24 antibody was assessed by western blot or in cells by immunofluorescence and confo- cal microscopic analysis.
Cell labeling and internalization assays
30 mm NH4Cl,
Cells grown on coverslips were washed twice with NaCl ⁄ Pi, fixed in 3% paraformaldehyde in NaCl ⁄ Pi for 30 min at 4 (cid:1)C and permeabilized with 0.1% Triton X-100 ⁄ 200 mm glycine in NaCl ⁄ Pi for 10 min at 4 (cid:1)C. Then, cells were washed with NaCl ⁄ Pi and exposed to secondary antibodies for 90 min at 37 (cid:1)C. Secondary antibodies were Alexa488- conjugated goat anti-mouse IgG (Santa Cruz Biotechno- logy, Santa Cruz, CA, USA) or rhodamine-conjugated goat anti-mouse IgG (Sigma-Aldrich, St Louis, MO, USA), both diluted 1 : 1000. After final washes with 1% BSA in NaCl ⁄ Pi, cells were mounted in FluorSave reagent (Calbio- chem, EMD Biosciences, La Jolla, CA, USA). Cells stained with LysoTracker or transferrin were fixed with 4% para- formaldehyde in NaCl ⁄ Pi for 30 min at room temperature, incubated with 50 mm NH4Cl for 10 min, permeabilized with 0.1% saponin ⁄ 2% BSA in NaCl ⁄ Pi and then proc- essed as indicated above.
Cells transiently transfected or not with plasmids indicated above were incubated on ice for 10 min to inhibit intra- cellular transport. Then, cells were incubated on ice for 45 min with hybridoma (ATCC No. HB-8445) supernatant containing R24 antibody in order to label GD3 ganglioside expressed on the cell surface. Afterwards, cells were washed three times with cold NaCl ⁄ Pi, transferred to 37 (cid:1)C with fresh prewarmed complete DMEM to allow antibody inter- nalization for different times, and finally harvested by scra- for ping or fixed in 3% paraformaldehyde in NaCl ⁄ Pi 30 min at 4 (cid:1)C. Where indicated, growth medium was sup- 60 lm chloroquine, plemented with 2 lgÆmL)1 BFA or 10 lm monensin. BFA and monensin were present for at least 15 min before R24 endocytosis was allowed and for different times after the temperature change. Control cells were supplemented with the same amount of vehicle. For transferrin internalization, cells were first incubated for 90 min in DMEM without fetal bovine serum, then incubated at 4 (cid:1)C in cold DMEM containing 10 lgÆmL)1 Alexa647-transferrin (Molecular Probes, Eugene, OR, USA) and R24 antibody for 45 min, and then trans- ferred to 37 (cid:1)C with prewarmed DMEM, without fetal bovine serum, supplemented with 10 lgÆmL)1 Alexa647- transferrin and processed at different times. For lysosome staining, cells were incubated in DMEM without fetal bovine serum supplemented with 0.2 lm acidotropic probe LysoTracker Red DND-99 (Molecular Probes) for 15 min at 37 (cid:1)C before fixation. Where indicated, noninternalized antibody remaining at the cell surface was removed by acid stripping with 0.5% acetic acid buffer, pH 3.0, containing 0.5 m NaCl for 1 min on ice.
Confocal immunofluorescence microscopy
Confocal images were collected using a Carl Zeiss LSM5 Pascal laser-scanning confocal microscope (Carl Zeiss, Jena, Germany) equipped with an argon ⁄ helium ⁄ neon laser and a 100 · 1.4 numerical aperture, oil immersion objective (Zeiss Plan-Apochromat). Single confocal sections of 0.7 lm were taken parallel to the coverslip (xy sections). Images were acquired and processed with the Zeiss lsm image software. Final images were compiled with adobe photoshop 7.0. The fluorescence micrographs shown in this manuscript are representative of at least three independent experiments.
R24 recycling assay
Cells from clone 2 (GD3-expresing CHO-K1 cells) or SK-Mel 28 cells were incubated for 10 min on ice to inhibit intracellular transport and then with the R24 antibody for 45 min on ice. Cells were then transferred to 37 (cid:1)C for 20 min to allow R24 endocytosis. Cell surface-bound anti- body was then removed by acid stripping at 4 (cid:1)C, and cells were extensively washed with cold NaCl ⁄ Pi. Then they were incubated at 37 (cid:1)C with 1 mL prewarmed fresh DMEM in order to restore transport of internalized antibody. Cells and culture medium were recovered at different times. Pro- teins from the culture medium were precipitated with chlo- roform ⁄ methanol (1 : 4, v ⁄ v) and resuspended in NaCl ⁄ Pi.
Proteins were resolved by electrophoresis in 12% polyacryl- amide gels under reducing or nonreducing conditions and then transferred electrophoretically to nitrocellulose mem- branes for 1 h at 300 mA. Protein bands in nitrocellulose
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Electrophoresis and western blotting
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Endocytic trafficking of an antibody to GD3
confocal microscopy and image analysis. R.I.B., P.M.C. and G.A.G. are recipients of CONICET (Argentina) Fellowships. J.L.D. is Career Investigator of CONICET (Argentina).
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This work was supported in part by Grants from Sec- retarı´a de Ciencia y Tecnologı´a (SECyT)-Universidad Nacional de Co´rdoba, Consejo Nacional de Investigaci- ones Cientı´ficas y Te´cnicas (CONICET, grant No. PIP 5151), Fundacio´n Antorchas (Grant No. 14116-112) and Agencia Nacional de Promocio´n Cientı´fica y Tec- nolo´gica (FONCYT, Grant No. 01-13522). We thank G. Schachner and S. Deza for technical assistance with cell culture, and C. Mas for excellent assistance with
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Supplementary material
is available
(P) and supernatant (SN)
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then incubated with the R24 antibody at 16 (cid:1)C for 1 h. Then cells were acid stripped to remove mem- brane-bound antibody, fixed (left panel) or transferred to 37 (cid:1)C and fixed at 30 min (right panel). The pres- ence of R24 analyzed by using Alexa488-conjugated goat anti-mouse IgG. Cell boundaries (white lines) are (B) GD3-expressing CHO-K1 cells were indicated. transiently transfected with GFP-Rab5 wild-type (GFP-Rab5wt). After 24 h, cells were incubated at 16 (cid:1)C for 15 min and then with R24 at 16 (cid:1)C for 1 h. Cells were then acid stripped to remove membrane- bound antibody and fixed. The presence of R24 was analyzed by using rhodamine-conjugated goat anti- mouse IgG (red). Expression of Rab5 was detected by the intrinsic fluorescence of GFP (green). Single confo- cal sections of 0.7 lm were taken parallel to the cover- slip. Insets in the merge panel show details at higher magnification. The upper inset shows a clear segrega- tion of R24 antibody and Rab5 in sorting endosomes. On the other hand, the lower inset shows endosomes where colocalization between R24 antibody and Rab5 is clearly visualized (yellow areas). Scale bars: 10 lm. Fig. S3. R24 antibody released into the culture med- ium after recycling is not associated with exosomes. CHO-K1 cells were incubated with R24 antibody for 45 min on ice. Afterwards, cells were allowed to intern- alize the antibody for 20 min at 37 (cid:1)C, and then the temperature was shifted again to 4 (cid:1)C. The cell surface was then stripped of any remaining antibody with acid wash (0 min). Then cells were incubated at 37 (cid:1)C to restore intracellular transport, and cells and culture medium were recovered at 30 and 60 min. The culture medium was then centrifuged at 100 000 g for 2 h, and fractions were the pellet recovered. The presence of R24 antibody was analyzed in all samples (cells, P and SN) by western blot as indic- ated in Experimental procedures. This material is available as part of the online article from http://www.blackwell-synergy.com The following supplementary material online: Fig. S1. Effect of pH on GD3 ganglioside–R24 anti- body association. CHO-K1 cells (GD3+) were incub- ated with R24 for 45 min at 4 (cid:1)C (To). After being washed, cells were incubated in NaCl ⁄ Pi solution at pH 7.4, 6.0 or 5.0 for 1 h at 4 (cid:1)C. Then cells and cul- ture medium (NaCl ⁄ Pi solution) were recovered, and the presence of R24 antibody in both samples was ana- lyzed by western blot as indicated in Experimental pro- cedures. Fig. S2. R24 antibody is endocytosed and accumulated in sorting endosomes at 16 (cid:1)C. (A) GD3-expressing CHO-K1 cells were incubated at 16 (cid:1)C for 15 min and
