ARTICLE |
Correspondence to: Günter Schwarzmann, Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany.
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Summary |
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A radioactive and biotin-labeled analogue of GM1 (biotinGM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotinGM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotinGM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotinGM1 was detectable in the form of patches (0.1 µm in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotinGM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotinGM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotinGM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotinGM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes. (J Histochem Cytochem 47:10051014, 1999)
Key Words: biotinGM1, endocytosis, immunoelectron microscopy, intralysosomal membranes, lipid transport
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Introduction |
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Gangliosides comprise a family of amphipathic molecules that contain a ceramide moiety as a lipophilic anchor embedded in the outer leaflet of plasma membranes and a sialo-oligosaccharide residue exposed towards the extracellular space (
Although biosynthesis and degradation of gangliosides are biochemically well characterized (
Because it is possible to insert exogenous gangliosides into the plasma membrane of cultured cells (
Immunocytochemistry of proteins is a well-established and extensively studied field (
In this study we examined the intracellular distribution of biotinGM1 that participated for many hours in the endocytic membrane flow. Because the exogenous gangliosides and their derivatives, such as biotinGM1, become components of the plasma membrane, they can be used as membrane tracers for electron microscopy. Moreover, the technique developed in the course of this study allows direct visualization at high spatial resolution of the distribution of individual biotin-labeled GM1 molecules.
We were able to show the incorporation of biotinGM1 into the plasma membrane, where it became detectable mainly in circumscribed patches and in caveola-like structures. Because late endosomes and lysosomes contain internal membrane structures in the form of vesicles or lipid lamellae (
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Materials and Methods |
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All mixtures of solvents are given in proportions of volumes, and this is therefore not further indicated.
Cell Culture
Monolayer cultures of human skin fibroblasts obtained from biopsies of a male infant were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Eggenstein, Germany) supplemented with 10% (v/v) fetal calf serum (FCS; Cytogen, Berlin, Germany). Cells were grown at 37C in a water-saturated atmosphere of 5% CO2 in air.
Antibodies
The following mouse monoclonal antibodies (MAbs) were used in this study: H4A3, which reacts with LAMP-1, and H5C6, which reacts with LIMP (both from Developmental Studies Hybridoma Bank; Baltimore, MD); and EGF-R1, which reacts with an extracellular domain of the EGF receptor (
Analysis of Biotin-GM1 Uptake and Metabolism in Cultured Fibroblasts
Incubation media containing biotinGM1 were prepared as described previously (
PLT Embedding in LR Gold and Immunolabeling
Human skin fibroblasts grown in 25-cm2 flasks were incubated with biotinGM1 as described above for 72 hr at 37C. In controls, the biotinGM1 was omitted. Cells were harvested by treatment with a solution of proteinase K (Merck) 0.05 mg/ml in PBS for 3 min on ice, pelleted, fixed with 4% formaldehyde in 0.2 M HEPES, pH 7.4, and postfixed with 1% OsO4 for 10 min at 4C. In control samples, OsO4 fixation was omitted. Pellets were embedded in LR Gold (Polysciences; Warrington, PA) after a progressive lowering of temperature (PLT) embedding protocol: dehydration in ethanol 50% for 45 min at 0C, 70% for 60 min at -20C, 90% for 60 min at -20C; infiltration in LR Gold/ethanol 1:1 for 30 min at -20C, 7:3 for 60 min at -20C, pure LR Gold for 60 min at -20C, LR Gold containing 0.5% benzil at -20C for 60 min, overnight, and for 1 hr. Polymerization was achieved with UV light for 48 hr at -20C and for 48 hr at room temperature. Immunolabeling of sections was performed with goat anti-biotin antibodies conjugated to 10-nm gold particles (1:100), co-incubated with mouse anti-LAMP-1 MAb (H4A3, 1:100), followed by 6-nm goat anti-mousegold (1:50), or co-incubated with rabbit anti-MPR antibodies (1:120), followed by 6-nm goat anti-rabbitgold (1:50). Controls included single labeling for each antibody and deletion of the primary antibody. Sections were postfixed with 2% glutaraldehyde in 0.2 M HEPES, stained with 2% uranyl acetate, and viewed at 80 kV with a Philips CM 120 electron microscope (Philips; Eindhoven, The Netherlands).
Cryoultramicrotomy and Immunolabeling
Human skin fibroblasts grown in 25-cm2 flasks were incubated with biotinGM1 as described above for 72 hr at 37C. For controls, biotinGM1 was omitted. Cells were harvested with proteinase K as described above, pelleted, and fixed with 4% formaldehyde and 0.1% glutaraldehyde in 0.2 M HEPES, pH 7.2. Pellets were infused with 50% polyvinylpyrrolidone (PVP-10,000; Sigma, Deisenhofen, Germany) and 1.15 M sucrose in 0.1 M HEPES (modified according to
Localization of the Biotin-labeled GM1 Analogue in Comparison to the EGF Receptor in EGF-stimulated Fibroblasts
Fibroblasts were incubated for 72 hr at 37C with biotin-labeled GM1 analogues at a concentration of 10 µM in DME containing 0.3% FCS. Cells were washed thoroughly and incubated with 100 nM EGF (Sigma) for 1 hr at 4C according to
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Results |
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Uptake and Metabolism of BiotinGM1 by Human Fibroblasts
For biochemical studies, fibroblasts were incubated with 10 µM biotinGM1 (Figure 1) in DME with 0.3% FCS for 72 hr and 120 hr at 37C. The same concentration of biotinGM1 was used for morphological studies. After careful washes, the cells were harvested as described in Materials and Methods and the lipids were extracted from the cell pellets. The amount of cell-associated biotinGM1 was 4.4 ± 0.5 nmol/mg protein at both time points, indicating saturation of ganglioside uptake. For analysis of metabolic products of biotinGM1, the extracted lipids were separated by TLC and radioactive lipids were visualized by exposure to X-ray-sensitive film. As shown in Figure 2, biotinGM1 has been degraded to the corresponding GM2 and GM3 derivatives. Their structures have been confirmed previously by digestion of biotinGM1 with the appropriate glycohydrolases (
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Immunolabeling of LR Gold Sections
After 72 hr of incubation with biotinGM1, monolayer cells were harvested with proteinase K and formaldehyde-fixed. Cell pellets were postfixed with OsO4 followed by embedding in LR Gold, a plastic resin suitable for immunolabeling. Both the OsO4 fixation and the embedding at low temperature should minimize lipid redistribution. Quantification of the amount of radioactivity in the dehydration medium revealed that at most 1015% of the cell-associated biotinGM1 was extracted during dehydration. In comparison to this, about 70% of cell-associated biotinGM1 was lost during conventional Epon embedding.
As shown in Figure 3, the biotinGM1 is visible on the plasma membrane, in caveolae (Figure 3A), and in coated pits (Figure 3B). Moreover, the biotinGM1 is detectable over the membranes of late endosomes and lysosomes, immunocytochemically identified by the use of antibodies specific for MPR (Figure 3C) or LAMP-1 (Figure 3D). Interestingly, the label for MPR and LAMP-1 is visible on the perimeter membrane, whereas the biotinGM1 is mainly localized on the intraendosomal/intralysosomal membranes. Because of the OsO4 fixation, the membranes show an excellent contrast. The antibiotin label was always found close to membranes, suggesting minimal or no lipid redistribution during the dehydration steps in the embedding procedure. As expected, the labeling efficiency of the antibodies against MPR and LAMP-1 was quite low because these, like many other antigens, are sensitive to OsO4 fixation. When OsO4 fixation of the samples was omitted, the labeling efficiency was higher but membranes showed a poor contrast and details of organelle morphology could hardly be observed (
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Immunolabeling of Cryosections
Fibroblasts were incubated with the biotinGM1 for 72 hr as described and harvested with proteinase K. Fixed pellets were infused overnight with buffered 50% polyvinylpyrrolidone in 1.15 M sucrose as a cryoprotectant and frozen in liquid nitrogen. Frozen sections were picked up with a mixture of methylcellulose and sucrose according to
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Double labeling with goat anti-biotin and antibodies against LIMP (Figure 4D) revealed that the organelles are late endosomes and lysosomes. They contain many internal structures, such as vesicles and multilamellar membranes, that bear biotinGM1. To find out whether these internal membranes are the place at which degradation of plasma membrane components, such as proteins and lipids, takes place, we analyzed the distribution of biotinGM1 in comparison to the EGF receptor in EGF-stimulated fibroblasts.
Co-localization of BiotinGM1 with the EGF Receptor in EGF-stimulated Fibroblasts by Immunoelectron Microscopy
The downregulation of the EGF receptor is a biochemically and morphologically well-characterized phenomenon and takes place in lysosomes (
For localization of the EGF receptor we used MAb (EGF-R1) that recognizes a determinant in the peptide portion of the extracellular domain of the EGF receptor without interfering with binding of EGF (
As shown in Figure 5, organelles are visible that are positive for both biotinGM1 and EGF receptor on LR Gold sections as well as cryosections. The labeling efficiency of the anti-EGF-R1 antibody on LR Gold sections (Figure 5A) was low, probably due to loss of antigenicity during OsO4 fixation. The internal membranes are labeled with anti-biotin antibodies, indicating biotinGM1, and the EGF receptor is detectable on the same structures. The cryosections show multivesicular bodies (Figure 5B) that bear label for biotinGM1 and the EGF receptor on their internal vesicles. These results indicate that both the biotinGM1 and the EGF receptor, after stimulation with EGF, were transported to internal membranes of late endosomes and lysosomes, indicating the same pathway of endocytosis.
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Discussion |
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This article describes the localization of an exogenously added biotin-labeled analogue of GM1 along the endocytic pathway after insertion into the plasma membrane of cultured fibroblasts. This approach provides a number of advantages. It allows, for the first time, analysis of the fate of an individual labeled ganglioside by direct visualization. Moreover, it is possible to use labeled exogenous glycosphingolipids as membrane tracers in endocytosis, which is not possible with metabolically labeled endogenous gangliosides. For an exogenous glycosphingolipid to become a true tracer for membrane lipids in endocytosis, it must be inserted into and must become a component of the lipid layer of the plasma membrane.
Insertion of BiotinGM1 into Cell Membranes
In aqueous solutions, gangliosides occur in the form of micelles, monomers, and oligomers in an equilibrium at concentrations above the CMC of about 10-9 M or less (
As demonstrated with spin-labeled ganglioside analogues (
The insertion into plasma membranes of gangliosides from their micellar state is a slow process. To incorporate significant amounts of the biotinGM1 into cells, it was necessary to apply prolonged incubation times at 37C. We could show that maximal incorporation into cells of biotinGM1 was reached after 72 hr. Further incubation did not result in increased incorporation.
Localization of BiotinGM1 by Immunoelectron Microscopy
Studies of the intracellular distribution of lipids with the high resolution of electron microscopy require methods that maintain membrane structure and composition. Therefore, particular care must be taken to preserve membrane structure, especially in the case of lipid-rich membranes that are often poorly fixed. Previous studies had shown that the localization of Forssman glycolipid in cryosections was not satisfactory because of nonspecific labeling (
Endocytic Pathway of BiotinGM1-labeled Membrane
Immunolabeling of LR Gold sections and cryosections revealed that the biotinGM1 is detectable at the plasma membrane and that it occurs in the form of patches. In addition, it appears to be concentrated in caveolae, with only sparse labeling of clathrin-coated pits or vesicles. This result is in agreement with the findings of
Moreover, biotinGM1 could be detected intracellularly over intraendosomal and intralysosomal membranes. This clearly demonstrates that these internal membranes are in part plasma membrane-derived and reach the lumen of late endosomes and lysosomes during endocytosis. A similar targeting has been demonstrated for the EGF receptor after binding of EGF (
The localization of biotinGM1 on internal membranes of late endosomes and lysosomes is in accordance with the distribution of endogenous GM1 (
To further investigate the properties and roles of individual glycosphingolipids in intracellular membrane transport, the synthesis of other biotin-labeled glycosphingolipid derivatives and the immunolocalization after their uptake by cells are of major interest.
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Acknowledgments |
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Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 284/B5 to GS).
We are grateful to Dr Bernd Hoflack, Institut Pasteur, Lille, France, for the generous gift of rabbit anti-MPR antibodies. WM wishes to thank Drs Gareth Griffiths and Paul Webster for helpful discussions during the EMBO course in Prague, 1997.
Received for publication November 12, 1998; accepted March 25, 1999.
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