Article |
Address correspondence to Dr. Richard E. Pagano, Mayo Clinic and Foundation, Guggenheim 621-C, 200 First Street, S.W., Rochester, MN 55905-0001. Tel.: (507) 284-8754. Fax: (507) 266-4413. E-mail: pagano.richard{at}mayo.edu
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Abstract |
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Key Words: endocytosis; caveolae; cholesterol; Eps15; lipid storage diseases
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Introduction |
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Using these methods, several fluorescent SL analogues and SL-binding toxins have been shown to be endocytosed by temperature- and energy-dependent processes (Koval and Pagano, 1989, 1990; Schwarzmann and Sandhoff, 1990; Hoekstra and Kok, 1992; Martin and Pagano, 1994). Recycling of fluorescent sphingomyelin (SM) (Koval and Pagano, 1989, 1990; Mayor et al., 1993) and glucosylceramide (GlcCer) (Kok et al., 1991, 1992) between intracellular membranes and the PM has been studied extensively in a number of cell types, including human skin fibroblasts, CHO cells, and polarized cells. In addition to recycling, internalized lipids may be specifically targeted to other intracellular compartments, such as late endosomes/lysosomes and the Golgi apparatus, and evidence for endocytic sorting of lipids between these compartments has been provided by several groups (Kok et al., 1991; Mukherjee et al., 1999; Puri et al., 1999). Evidence that some SLs are targeted to the Golgi apparatus after endocytosis comes from the use of biotinylated, fluorescent, or nondegradable SL analogues, or the labeled B-subunits of cholera toxin (CtxB) or shiga toxin (StxB) which bind to GM1 ganglioside and globoside, respectively (Schwarzmann and Sandhoff, 1990; Schwarzmann et al., 1995; Chen et al., 1998; Puri et al., 1999; Grimmer et al., 2000; Sandvig and van Deurs, 2000).
The relative importance of specific mechanisms for the endocytosis and intracellular targeting of PM SLs are not known. Studies using StxB bound to the cell surface show that this lipidtoxin complex is internalized via clathrin-dependent endocytosis (Johannes and Goud, 1998). In addition, a fluorescent analogue of SM partially colocalizes with endocytosed transferrin receptor within seconds of internalization from the PM, suggesting that at least a portion of the SM analogue is endocytosed via the clathrin pathway (Chen et al., 1997). A second potential mechanism for endocytosis of SLs is internalization via caveolae. Endocytosis through caveolae has been best characterized as a mechanism for the entry of small molecules such as folic acid; however, CtxB bound to GM1 ganglioside, endothelin and growth hormone receptors, antibodycross-linked alkaline phosphatase, SV-40 virus, and bacteria have all been reported to be internalized via caveolae or "caveolar-like" processes (Parton, 1994; Orlandi and Fishman, 1998; Chen and Norkin, 1999; Lobie et al., 1999; Okamoto et al., 2000; Shin et al., 2000; Pelkmans et al., 2001). Finally, in addition to the clathrin and caveolar endocytic mechanisms, caveolar-like endocytosis is exhibited by certain cell types that lack caveolin-1, and some reports suggest the existence of additional nonclathrin, noncaveolar endocytic processes in some cells (Orlandi and Fishman, 1998; Sandvig and van Deurs, 2000).
In the current study, we examine the mechanism of endocytosis of several different SL analogues from the PM of human skin fibroblasts and provide evidence that two glycosphingolipid (GSL) analogues were selectively internalized via a clathrin-independent pathway, whereas another SL, SM, was internalized by both clathrin-dependent and -independent mechanisms. Furthermore, we show that although both internalization pathways led to Golgi targeting of SLs in normal cells, the initial mode of internalization was a major factor in determining the subsequent targeting of the SLs to late endosomes/lysosomes versus the Golgi apparatus in SL storage disease (SLSD) fibroblasts. These findings were confirmed using SL binding toxins which monitor the trafficking of endogenous SLs at the PM, demonstrating that our results were not limited to fluorescent SL analogues.
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Results |
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We next studied the fate of BODIPY-LacCer and -SM present in "30 s endosomes" after various periods of further chase at 37°C (Fig. 2) . There were no obvious differences in the number or distribution of endosomes formed from LacCer versus SM after the initial 30 s of internalization. However, at this time point virtually all of the endosomes appeared green in color when LacCer was used, whereas in the case of SM, numerous red/orange endosomes (indicative of high BODIPY-SM concentrations) as well as green endosomes were seen (Fig. 2 a, 30"/0"). After further incubation at 37°C in the absence of metabolic inhibitors, there was no obvious qualitative change in the appearance of the SM-containing endosomes during 1060 s of chase (Fig. 2 a). However, for LacCer, many red/orange endosomes were observed at 10 s, but at later times of chase all endosomes became green in color (Fig. 2 a). Analogous experiments were carried out using BODIPY-globoside, which behaved similarly to the LacCer analogue (data not shown). The changes in R/G ratios of SM- and LacCer-containing endosomes over time are also shown quantitatively (Fig. 2 b). These results show that LacCer, but not SM, was transiently concentrated in early endosomes and suggested that the BODIPY-LacCer and -SM analogues might be internalized by different mechanisms and/or sorted differently after internalization.
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Internalization and subsequent targeting of SL analogues in storage disease fibroblasts and in normal cells overloaded with cholesterol
BODIPY-LacCer is internalized from the PM and transported predominantly to the Golgi complex of normal cells, whereas in numerous SLSD cell types, the LacCer analogue accumulates in punctate cytoplasmic vesicles which partially colocalize with fluorescent dextran and lysotracker dyes as a result of elevated intracellular cholesterol (Chen et al., 1998, 1999; Puri et al., 1999; unpublished data). Therefore, it was of interest to learn whether the initial internalization mechanism for LacCer was altered in SLSD cells. We found that pretreatment of SLSD cells with mß-CD followed by pulse-labeling with the LacCer analogue eliminated nearly all intracellular labeling, whereas chlorpromazine had no effect using Niemann Pick Type C (NP-C) (Fig. 6
a), Niemann Pick Type A, GM1- or GM2-gangliodosis cells (data not shown). Transfection with dominant negative Eps15 also had no effect on LacCer internalization or targeting in these cell types (data not shown). Thus, BODIPY-LacCer was internalized via a chlorpromazine- and Eps15-insensitive mechanism in SLSD cells as well as in normal cells, even though Golgi targeting is disrupted in these cells. Furthermore, BODIPY-globoside was also targeted to endosomes/lysosomes in SLSD cells and this internalization was similarly mß-CD-sensitive and chlorpromazine-insensitive (data not shown).
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In parallel experiments, we also studied the trafficking of BODIPY-SM and -LacCer in normal fibroblasts after overloading the cells with cholesterol (Fig. 6 b). In control fibroblasts with normal levels of cholesterol, both the SM and LacCer analogues were targeted primarily to the Golgi apparatus, although some punctate cytoplasmic fluorescence was also seen. In contrast, when cells were grown under conditions which elevated cellular cholesterol (Puri et al., 1999), Golgi targeting of LacCer was eliminated and almost all the lipid was targeted to punctate cytoplasmic structures. In the case of SM, overloading with cholesterol did not eliminate Golgi targeting, but the punctate cytoplasmic staining was much more prominent than in control cells (Fig. 6 b, XS-Chol). Chlorpromazine had no effect on LacCer targeting in cholesterol-overloaded cells, whereas for the SM analogue, punctate cytoplasmic labeling without any obvious Golgi fluorescence was now observed (Fig. 6 b, XS-Chol + chlorpromazine). In control experiments with normal cells, chlorpromazine did not affect the Golgi targeting of BODIPY-SM or -LacCer internalized through the clathrin-independent pathway (data not shown). These results show that SL uptake through the clathrin pathway gave rise to Golgi labeling in all cell models tested, whereas SLs which enter by the clathrin-independent route are subject to cholesterol-modulated intracellular targeting in both normal and SLSD fibroblasts.
Finally, we studied the internalization and targeting of endogenous PM SLs in control and SLSD fibroblasts using fluorescently labeled SL-binding toxins. For these experiments we used CtxB, which binds to GM1 ganglioside and is internalized via caveolae or a caveolar-like mechanism (Orlandi and Fishman, 1998), and StxB, which binds globosides and is internalized by clathrin-mediated endocytosis (Sandvig and van Deurs, 1994; Johannes and Goud, 1998). As shown in Fig. 7 , CtxB was targeted to the Golgi apparatus of normal cells, but to punctate structures in GM1 gangliosidosis fibroblasts. Importantly, we found that cholesterol depletion of GM1 gangliosidosis fibroblasts restored the targeting of CtxB to the Golgi complex of these cells (Fig. 7), similar to the effect of cholesterol depletion on BODIPY-LacCer targeting in SLSD cells (Puri et al., 1999). In contrast, StxB was transported to the Golgi complex of both normal and GM1 gangliosidosis cells regardless of the level of cellular cholesterol. Similar results were obtained using these toxins in GM2 gangliosidosis, NP-A, and NP-C fibroblasts (data not shown).
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Discussion |
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LacCer and globoside, but not SM, are internalized almost exclusively by a clathrin-independent mechanism
Our results show that BODIPY-LacCer and -globoside were internalized from the PM of human skin fibroblasts exclusively by a clathrin-independent mechanism. Several lines of evidence strongly suggest that this clathrin-independent mechanism involved internalization through caveolae. First, we showed that inhibitors of caveolar-like endocytosis (e.g., of CtxB) completely blocked internalization of LacCer, whereas inhibitors of clathrin-dependent endocytosis had no significant effect on LacCer internalization. In contrast, uptake of SM was partially inhibited by both classes of inhibitors. To rule out nonspecific effects of the drug treatments, we demonstrated similar results using multiple inhibitors, which inhibit endocytosis by different mechanisms. Second, we demonstrated that the internalization of LacCer and SM were both inhibited by expression of a dominant negative dynamin mutant. Inhibition of internalization of both lipids is consistent with our interpretation of the endocytic mechanisms involved in SL uptake, since dynamin is reported to be essential for both clathrin-dependent and caveolar uptake. Expression of an Eps15 dominant negative mutant affected only the internalization of SM, consistent with our results showing partial inhibition of SM, but not LacCer, uptake by inhibitors of the clathrin pathway.
To provide further characterization of SL uptake through the clathrin-independent pathway, we showed extensive colocalization of BODIPY-LacCer with Rh-CtxB and of Rh-CtxB with caveolin-1GFP (Figs. 4 and 5). These experiments suggest that (a) CtxB marks the caveolar compartment in human skin fibroblasts, as shown previously in other cell types (Parton, 1994), and (b) most of the intracellular LacCer analogue was present in a caveolin-1positive compartment after 5 min of internalization. However, we note that transfection of human skin fibroblasts with an NH2-terminal GFP construct of caveolin-1 had no effect on internalization of the SL analogues in the present study (data not shown) although this construct blocked internalization of SV-40 via the caveolar pathway in CV-1 cells (Pelkmans et al., 2001). In contrast to the LacCer analogue, we found partial overlap of SM with both Rh-CtxB and DiI-LDL in colocalization studies (Fig. 5). This result fully supports our conclusion that SM is endocytosed approximately equally by both pathways.
Finally, we note that the initial pathways of internalization for the SL analogues were the same in SLSD cells as in normal fibroblasts, since the uptake of LacCer and globoside analogues was nearly completely inhibited by mß-CD in both normal (Table I) and SLSD cells (Fig. 6 a), whereas SM uptake was only partially inhibited by the different classes of inhibitors (Table I; Fig. 6) or by expression of dominant negative Eps15 (data not shown).
Initial mechanisms of SL internalization determine the utilization of two distinct Golgi targeting pathways
Our studies have further defined the pathways by which PM SLs are internalized and transported to the Golgi apparatus versus the late endosomes and lysosomes (Fig. 8)
. When SLSD cells were pulse labeled with BODIPY-SM, the fluorescent lipid entered cells by a combination of clathrin-dependent and -independent internalization mechanisms. These dual pathways of uptake gave rise to both punctate cytoplasmic labeling and to Golgi labeling as seen in Fig. 6 a. The "Golgi component" of this fluorescence could be eliminated by blocking uptake through the clathrin pathway (e.g., by chlorpromazine treatment or by expression of dominant negative Eps15), whereas the "punctate cytoplasmic" component could be eliminated by inhibition of the clathrin-independent internalization (i.e., by mß-CD treatment) (Fig. 6 a, right versus middle panels for SM).
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Together, these results demonstrate that the initial pathways of SL internalization define two discrete Golgi targeting pathways as outlined in Fig. 8. We are currently further characterizing these two pathways and have recently found that SLs internalized by the clathrin-independent, caveolar-like pathway reach a late endosomal compartment by a rab7-dependent step before being transported to the Golgi apparatus, whereas SLs internalized via the clathrin-mediated pathway are targeted to the Golgi apparatus independent of rab7 (unpublished data).
In summary, our studies provide convincing evidence for the existence of two independent pathways for internalization and Golgi targeting of PM SLs; however, many important questions remain concerning these transport pathways. (a) The mechanism for sorting of SL analogues into clathrin-coated pits versus caveolar-like PM specializations is unknown. Although we were able to observe differential sorting and enrichment of LacCer and SM analogues in endocytic vesicles derived from different PM specializations within the first 3040 s of endocytosis (Fig. 2), we were unable to visualize a heterogeneous distribution of these analogues at the PM before internalization (Fig. 1). The absence of such heterogeneities, comparable in size to early endosomes, suggests that any PM "microdomains" were either too small to be visualized (Kenworthy et al., 2000; Pralle et al., 2000) by our current methods or an alternative mechanism for sorting of PM SLs needs to be considered. (b) The itinerary for the internalized SLs and the determinants for Golgi complex targeting need to be identified. The demonstration that only the targeting of SLs internalized via the caveolar-like pathway was perturbed by cholesterol suggests that SLs internalized by different mechanisms may be transported through two different sets of endosomal compartments, only one of which is sensitive to intracellular cholesterol. Further studies are required to define these intracellular compartments and to determine the site and mechanism by which cholesterol modulates targeting. The existence of two endosomal pathways for Golgi complex targeting of SLs also raises the question of where along the endosomal pathway do components of vesicles derived from the clathrin-dependent and -independent pathways intermix (Tran et al., 1987) with one another. As depicted in Fig. 8, it is possible that SLs internalized by the two pathways are always in different endosomal vesicles and never intermix, or alternatively intermix at some point along the endocytic pathway, and then subsequently segregate into discrete pathways with unique characteristics with respect to cholesterol modulation. (c) The functional significance of the two PM-to-Golgi complex pathways needs to be determined. Presumably, the delivery of SLs to the Golgi complex by two independent routes represents part of the overall recycling pathway for PM SLs. The possible internalization of LacCer via caveolae and the modulation of LacCer targeting to the Golgi complex by cellular cholesterol suggest a novel role for sterol in regulating lipid recycling. These data also raise the possibility that (some) cholesterol may be transported to the Golgi complex (in normal cells) or retargeted to endosomes/lysosomes (in SLSD cells) via the same pathway that we have identified for the LacCer and globoside analogues.
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Materials and methods |
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Fluorescent lipids, toxins, and other reagents
BODIPY-SM, BODIPY-Ceramide, and Lysotracker Red were from Molecular Probes. BODIPY-LacCer was synthesized and purified as described (Martin and Pagano, 1994); BODIPY-globoside was synthesized in an analogous manner using lyso-ceramide trihexoside (Matreya, Inc.). Complexes of the BODIPY-SLs with defatted bovine serum albumin (DF-BSA) were prepared as described (Martin and Pagano, 1994) and diluted in HMEM (10 mM Hepes-buffered minimal essential medium, pH 7.4). Rh-labeled CtxB was from List Biological Laboratories, Inc. FITC-labeled StxB was a gift from Dr. David Haslam (Washington University School Medicine, St. Louis, MO). DiI-LDL was from Intracel. All other reagents were from Sigma-Aldrich unless otherwise noted.
Incubation of cells with BODIPY lipids
Cell cultures were washed with ice cold HMEM, transferred to a water bath at 10°C, and then incubated with varying amounts of BODIPY-SL/DF-BSA for 30 min to label the PM (see Results). The cells were then washed with cold HMEM and warmed to 37°C for various times to induce endocytosis. After this incubation, the medium was replaced with ice cold HMEM without glucose containing the metabolic inhibitors, 5 mM NaN3, 50 mM 2-deoxyglucose, and 10 µM CCCP (HMEM-G+I), and the culture dishes were transferred to a 10°C bath. Fluorescent lipid present at the cell surface was then removed by "back exchange" (6 x 10 min incubations with 5% DF-BSA in HMEM-G+I at 10°C [Chen et al., 1998]). The cells were then observed under the fluorescence microscope using a temperature-controlled stage maintained at 4°C.
Inhibitor treatments
Human skin fibroblasts grown on glass coverslips were treated with inhibitors of endocytosis as follows. (a) Chlorpromazine (Gustavsson et al., 1999; Okamoto et al., 2000): samples were incubated with 6 µg/ml of the drug for 30 min at 37°C and then pulse labeled with fluorescent lipid in the presence of chlorpromazine. (b) Potassium depletion (Larkin et al., 1983; Hansen et al., 1993): samples were rinsed with K+-free buffer (140 mM NaCl, 20 mM Hepes, 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml D-glucose, pH 7.4) and then rinsed in hypotonic buffer (K+-free buffer diluted 1:1 with distilled water) for 5 min. Cells were then quickly washed three times in K+-free buffer and incubated for 20 min at 37°C in K+-free buffer. Samples were then incubated with fluorescent lipids as above, except that all solutions were K+-free. Control experiments were carried out in an identical manner, except that all solutions contained 10 mM KCl. (c) mß-CD (Hansen et al., 2000; Okamoto et al., 2000): cells were pretreated with 10 mM mß-CD in serum-free EMEM for 30 min at 37°C to deplete PM cholesterol before incubation with fluorescent lipid. In separate experiments, cellular cholesterol was quantified (Slotte and Bierman, 1988) and found to be 25% depleted by mß-CD treatment. (d) Nystatin (Rothberg et al., 1992): samples were incubated with 25 µg/ml nystatin in serum-free EMEM for 30 min at 37°C, washed with HMEM containing 25 µg/ml nystatin, and then pulse labeled with the fluorescent lipids in the presence of nystatin. (e) Genistein (Aoki et al., 1999; Chen and Norkin, 1999; Liu and Anderson, 1999): samples were pretreated with 200 µM genistein for 2 h in HMEM, washed, and pulse labeled with the fluorescent lipids as above except with 200 µM genistein present throughout. No significant difference in PM loading of the fluorescent SL analogues was observed in inhibitor-treated versus -untreated cells. For each inhibitor treatment, cell viability was >90%; quantitative lipid analyses (Chen et al., 1998) also showed that the degradation of the SM and LacCer analogues was <10%.
Transfection studies
The following GFP-constructs in mammalian expression vectors were gifts as indicated: Eps15 (D32 [control] and the EH21 mutant E
95/295 [dominant negative Eps15]) were from Drs. A. Benmerah and A. Dautry-Varsat (Institut Pasteur, Paris, France); dynamin 2 (Dyn2ab [control] and GFP-Dyn2ab K44A [dominant negative]) were from Dr. M. McNiven (Mayo Clinic); NH2- and COOH-terminal GFP-constructs of caveolin-1 were from Drs. L. Pelkmans and A. Helenius (Swiss Federal Institute of Technology) and Dr. R.G.W. Anderson (University of Texas Southwestern, Dallas, TX). Cells were treated with TransIT-LT1 transfection reagent (Mirus Corporation) and 2 µg/ml DNA using the manufacturer's protocol. After a 46-h treatment, the cells were washed and subsequently cultured for 1824 h in EMEM containing 10% FBS before treatment with the fluorescent lipids as above. Transfected cells were detected by GFP-fluorescence and the effect on lipid internalization was evaluated from observations in the red region of the spectrum where BODIPY-lipid, but not GFP-fluorescence, could be visualized.
Microscopy and colocalization studies
Conventional fluorescence microscopy was performed with an fluorescence microscope (IX70; Olympus) equipped with filter packs which allowed the specimens to be excited at 450490 nm and viewed at "green" (em = 520-560 nm), "red" (
em = 590 nm), or "green + red" (
em
520 nm) wavelengths (Pagano et al., 1991). Rh-CT and DiI-LDLlabeled specimens were observed under the fluorescence microscope using optics appropriate for these fluorophores (
ex = 540/25 nm;
em = 620/60 nm). In any given experiment, all photomicrographs were exposed and printed identically. Confocal microscopy was carried out using a ZEISS model 510 instrument using a 100x (1.4 NA) objective. Quantitative image analysis was performed using the "Metamorph" image processing program (Universal Imaging Corp.) as described (Chen et al., 1997; Pagano et al., 2000).
For colocalization studies using BODIPY-SLs, cells were incubated for 30 min at 10°C with the BODIPY-lipid (see above) in the presence of 0.2 µM Rh-CtxB or 0.5 µg/ml DiI-LDL (Molecular Probes, Inc.) and washed. The samples were then incubated for 30 s at 37°C and back exchanged. (For DiI-LDL treated specimens, the samples were then washed with HMEM-G+I and "acid stripped" to remove any DiI-LDL remaining at the cell surface [Hopkins and Trowbridge, 1983]). Samples were then chased at 37°C and observed under the fluorescence microscope. In control experiments using cells in which only Rh-CtxB or DiI-LDL was present, no fluorescence was detected with the "green" or "red" microscope filters under the exposure conditions used for BODIPY-fluorescence.
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Footnotes |
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Rikio Watanabe's present address is Niigata University School of Medicine, Dermatology Department, Niigata 951, Japan.
Michel Dominguez's present address is Caprion Pharmaceuticals, H4P 1P7 Montreal, Canada.
* Abbreviations used in this paper: BODIPY, boron dipyrromethenedifluoride; CtxB, cholera toxin, B-subunit; DF-BSA, defatted bovine serum albumin; Dyn2, dynamin 2; Eps15, EGFR pathway substrate clone 15; GSL, glycosphingolipid; LacCer, lactosylceramide; mß-CD, methyl-ß-cyclodextrin; PM, plasma membrane; SL, sphingolipid; SLSD, sphingolipid storage disease; SM, sphingomyelin; StxB, shiga toxin, B-subunit.
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Acknowledgments |
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This work was supported by grants from the United States Public Health Service (GM-22942 and GM-60934) and the Ara Parseghian Medical Research Foundation to R.E. Pagano, a Kendall-Mayo Fellowship to M. Dominguez, and an American Heart Association Fellowship to V. Puri.
Submitted: 15 February 2001
Revised: 26 June 2001
Accepted: 3 July 2001
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