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First published online 21 April 2009
doi: 10.1242/jcs.039727

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Endocytosis of MHC molecules by distinct membrane rafts

Department of Cell Biology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

View larger version (48K): [in this window] [in a new window]   Fig. 1. Internalization of MHC I and MHC II by distinct endocytic carriers in M12.C3F6 cells. Monoclonal antibodies (MAbs) bound to MHC I (R1-9.6) and MHC II (40F) at the cell surface were internalized at 37°C for the indicated times and detected by cross-adsorbed secondary antibodies after fixation and permeabilization. Representative equatorial sections from confocal stacks were selected (rows 1-3). Note that most internalized MHC I and MHC II localized to distinct peripheral structures after 5 minutes, but merged in perinuclear endosomes at 10-20 minutes. (Bottom row) Steady state distribution of MHC proteins in fixed M12.C3.F6 cells. Scale bars: 2 µm. (Left) The staining pattern of endocytic vesicles from 20 equatorial sections per condition was recorded and the percentage of single-positive endocytic vesicles plotted against the endocytosis time. Error bars denote s.d.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Co-internalization of MHC molecules with cholera toxin B (CTB). M12.C3F6 cells were labeled with CTB-FITC and either mAb 40F (rows 1-2) or mAb R1-9.6 (rows 3-4) on ice, then warmed to 37°C for 5 or 20 minutes, and stained with secondary reagents after fixation and permeabilization. Rows 1-4 show representative equatorial sections from confocal stacks. (Left) The percentage of MHC-containing vesicles that stained only for MHC and not for CTB-FITC (`MHC-only') is plotted. Per condition 20 equatorial sections were analyzed, error bars denote s.d. Note that most MHC II-positive endocytic vesicles contained CTB at 5 and 20 minutes of endocytosis, whereas MHC I significantly colocalized with CTB only at 20 minutes of endocytosis, and not at 5 minutes (60% `MHC I-only' vesicles). Also note the presence of many CTB-containing vesicles devoid of MHC II in the top row. Scale bars: 2 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 3. Cholesterol-dependent endocytosis of MHC proteins. M12.C3.F6 cells were pre-treated or not with 15 µM filipin III before surface biotinylation and internalization in the presence or absence of filipin. Surface biotin was removed by GSH (right) or not (left) and MHC I and MHC II were sequentially immunoprecipitated and probed for biotin (top and middle panel). The remaining biotinylated proteins were retrieved and probed for TfnR (lower panel). MHC proteins were quantified as described in Fig. S3 (see supplementary material), TfnR was quantified by densitometry. Internalization was expressed relative to the mean of samples not stripped by GSH. Shown are results of one experiment out of two with similar results. Note that internalization of MHC proteins was slow and sensitive to filipin, whereas endocytosis of TfnR was fast and almost unaffected by filipin.

View larger version (78K): [in this window] [in a new window]   Fig. 4. Analysis of the membrane environment of patched MHC proteins in M12.C3.F6 cells. Clusters of MHC I (A) or MHC II (B) on M12.C3.F6 cells were induced with mAbs R1-9.6 (A) or 40F (B) and appropriate secondary reagents. GM1 was patched by CTB-FITC. Clusters of MHC II and MHC I (C) or B220 (D) were induced with mAb 40F and mAbs R1-9.6 (C) or RA3-6B2 (D), and cross-adsorbed secondary reagents. Note the extensive co-patching of MHC II and GM1 (B), the low co-patching of MHC I and GM1(A), or MHC I and MHC II (C), and its absence from the MHC II-B220 sample (D). Scale bars: 2 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Separation of MHC I- and MHC II-containing DRMs. (A) Brief Brij 98 extraction following a protocol derived from Drevot et al. (Drevot et al., 2002). (B) Rigorous Brij 98 extraction protocol. (C) Flotation of surface-biotinylated MHC I and MHC II molecules after rigorous Brij 98 extraction. Note that MHC I and MHC II floated to separate fractions in B and C, but not in A. Also note that MHC I- and MHC II-containing DRMs were both derived from the same membrane, the plasma membrane, in C. AP, alkaline phosphatase; sMHC II ,β, cell surface MHC II subunits; sMHC I HC, cell surface MHC I heavy chain; sMHC I LC, cell surface MHC I light chain; rel. distr., relative distribution.

View larger version (67K): [in this window] [in a new window]   Fig. 6. Polypeptide and lipid composition of dense and light DRMs of M12.C3.F6 cells. (A) DRMs prepared by rigorous Brij 98 extraction at a detergent to protein ratio of 1.25. (Top) Marker profiles. (Bottom left) Protein profile by silver staining. (Bottom right) Coomassie-stained gel of dense and light DRMs, each 33-fold concentrated, and soluble protein (no further concentration). (B) Lipid composition of PNMs and dense and light DRMs. Lipids were extracted from PNMs or from pelleted DRMs, separated by HPTLC, stained with primulin (left), and quantified (right, five independent experiments). The relative lipid content was calculated assuming identical molar primulin adsorption of all lipids. SM and PI were quantified together. Error bars denote s.d. Note the abundance of cholesterol, SM/PI and PS in light DRMs, and of PE in dense DRMs. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin.

View larger version (66K): [in this window] [in a new window]   Fig. 7. Sensitivity of dense and light DRMs to cholesterol extraction. M12.C3.F6 cells were extracted with 10 mM MCD or left untreated before PNM preparation and rigorous extraction with Brij 98. In addition to individual proteins, total protein was analyzed by silver staining. Gels from control and MCD-treated samples were stained in one tray for comparability. Note that MHC I, MHC II and light DRM polypeptides were strongly diminished at their DRM position by MCD treatment, whereas polypeptides associated with dense DRMs were only slightly affected. AP, alkaline phosphatase; con, control; MCD, β-methyl-cyclodextrin.

View larger version (74K): [in this window] [in a new window]   Fig. 8. Distribution of GM2 to MHC I- and MHC II-containing membrane environments. (A) Localization of GM2 in dense and light DRMs. Immunoblot analysis of the distribution of MHC I, MHC II, GM1 and GM2 after rigorous Brij 98 extraction and flotation. Owing to low GM2 levels in M12.C3.F6 cells, DRMs were concentrated 20-fold before dot blotting with anti-GM2. (B) Co-clustering analysis of GM2 and MHC proteins. GM2 and MHC I or MHC II were clustered by primary and cross-adsorbed secondary antibodies. Only tangential confocal sections are displayed. Note that both MHC I and MHC II showed co-clustering with patched GM2. Scale bars: 2 µm.

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