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First published online 26 February 2008
doi: 10.1242/jcs.022731

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Distinct endocytic recycling of myelin proteins promotes oligodendroglial membrane remodeling

Department of Biology, Unit of Molecular Cell Biology, University of Mainz, Bentzelweg 3, 55128 Mainz, Germany

View larger version (79K): [in this window] [in a new window]   Fig. 1. Localization of myelin proteins in endocytic compartments. Primary oligodendrocytes differentiated for 4 days in culture were incubated with transferrin-FITC (green) to label recycling endosomes. Cells were stained using antibodies specific for the myelin proteins PLP, MAG or MOG (red), and antibodies against the LE/Lys marker LAMP1 (blue). Localization of myelin proteins in the LE/Lys appears magenta and localization in the recycling endosomes appears yellow. Confocal planes are shown, insets show enlarged areas. More than 50 cells were analyzed per condition in each of 3-5 experiments. Scale bars: 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Endocytic uptake of myelin proteins in oligodendrocytes. (A) Oli-neu cells transiently expressing the myelin proteins PLP, MAG or MOG (a) and primary cultured oligodendrocytes (b) were cell-surface biotinylated using a reducible biotin analogue. Cells were either lysed immediately after biotinylation (lane 1, total biotinylation), kept on ice to block endocytosis followed by cleavage of biotin with the reducing agent DTT (lane 2, control), or incubated at 37°C to allow endocytosis followed by biotin cleavage (lane 3). After cell lysis, biotinylated proteins were precipitated with NeutrAvidin beads and analyzed by western blotting. Endocytosed PLP, MAG and MOG, but not Na+/K+-ATPase, were recovered from cells, which were subjected to endocytosis and biotin cleavage (lane 3). Densitometric quantification of endocytosis of biotinylated proteins of 3-5 independent experiments was performed with primary cultured oligodendrocytes (c). Error bars indicate s.e.m.; *P<0.05 (paired t-test). (B) Antibody-uptake experiments using Oli-neu cells transiently expressing the myelin proteins PLP, MAG or MOG and cells expressing endogenous NG2. Live cells were incubated on ice with primary antibodies recognizing cell-surface epitopes of PLP, MAG, MOG or NG2 followed by Cy3-coupled secondary antibodies. After endocytosis for 0 minutes, 15 minutes or 1 hour at 37°C, surface-localized antibody complexes were counterstained with Cy2-coupled antibodies. Endocytosed proteins appear red, cell-surface-localized proteins appear yellow. Internalized MOG localizes to the perinuclear region (arrowheads). Scale bars: 10 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Differential endocytic targeting of PLP, MAG and MOG. (A) Colabeling of endocytosed proteins with endocytic markers. Transfected Oli-neu cells were allowed to endocytose surface-bound antibodies for 1 hour at 37°C. During the endocytosis period, labeling of recycling endosomes was achieved by co-endocytosis of transferrin-FITC (bottom images, green). To label LE/Lys, cells were immunostained using anti-LAMP1 antibodies (top images, green). Confocal images show colocalization of PLP and MAG with LAMP1 in LE/Lys (top images, yellow). MOG colocalized with transferrin-FITC in the recycling endosomes (bottom image, yellow). (B) Co-endocytosis of PLP (red) and MAG (green), performed by simultaneous antibody uptake. Cell-surface-localized proteins were counterstained in blue and thus appear cyan, magenta or white. After 5 minutes of endocytosis PLP and MAG are localized to distinct populations of early endosomes (red and green dots). Colocalization of endocytosed PLP and MAG increases over 10 and 30 minutes of endocytosis (yellow dots). Insets show enlarged areas. Scale bars: 5 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Analysis of clathrin-dependent endocytosis. The clathrin-sequestering protein AP180C-myc was overexpressed in Oli-neu cells to interfere with clathrin-mediated endocytosis. AP180C-expressing cells were identified by staining with anti-myc antibodies (top row). Color images (bottom row) depict endocytosis (red) and cell surface localization (green/yellow). The graphs show the percentage of endocytosing AP180C-negative cells (black bars, control) and the percentage of endocytosing AP180C-expressing cells (gray bars). (A) The clathrin-mediated uptake of transferrin Alexa Fluor 594 (red) is blocked specifically in AP180C-overexpressing Oli-neu cells (arrowheads). Blue color depicts DAPI-stained nuclei. (B) Coexpression of AP180C-myc and PLP, MAG or MOG. Transfected cells were subjected to antibody-uptake experiments as described in Fig. 2B. Stacked confocal images of representative cells are shown. Overexpression of AP180C strongly reduced the internalization of MAG and MOG. More than 50 cells were counted per three (PLP and MAG) or four (MOG) independent experiments. Error bars represent s.e.m.; **P<0.01; ***P<0.001 (paired t-test). Scale bars: 5 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Analysis of clathrin-independent endocytosis. To inhibit clathrin-independent cholesterol-dependent endocytosis, Oli-neu cells transiently expressing PLP, MAG and MOG were treated with the cholesterol-depleting agent filipin (1 µg/ml) during antibody uptake. To exclude that cholesterol depletion affects clathrin-dependent endocytosis, transferrin-FITC (green) was co-endocytosed with the myelin proteins and cells internalizing transferrin were exclusively analyzed. Cell-surface PLP, MAG or MOG was counterstained in blue. In merged images, surface-localized proteins thus appear magenta, whereas endocytosed proteins appear red (or yellow in case of colocalization with transferrin-FITC). Confocal images of representative cells are shown. The percentage of cells internalizing PLP, MAG or MOG was calculated from the population of transferrin-internalizing cells (graphs: black bars, untreated cells; grey bars, filipin-treated cells). More than 50 cells were counted per three (PLP and MAG) or five (MOG) independent experiments. PLP endocytosis was inhibited by filipin treatment, whereas endocytosis of MAG and MOG was unaffected. Error bars represent s.e.m.; **P<0.01 (paired t-test). Scale bars: 5 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 6. Cultured oligodendrocytes develop morphologically and biochemically distinct membrane domains. (A) Confocal analysis of co-stained primary oligodendrocytes demonstrates association of PLP (green), MAG (red, top images) and MOG (red, bottom images) with distinct membrane domains. Dashed circles delineate cell bodies and boxed areas are enlarged in a' and a''. Asterisks indicate membrane sheets. Arrows indicate the typical localization of MAG (a') and MOG (a''). Scale bars: 5 µm. (B) Subfractionation of myelin and oligodendroglial membranes by discontinuous density gradient centrifugation in heavy (H), medium (M) and light (L) membrane fractions, analyzed by western blotting (nonreducing conditions, thus the dimeric form of PLP is prominent). Myelin was isolated from postnatal day 10 (P10) mouse brain at a developmental stage analogous to that of the cells in culture. Note that the compact myelin domain (indicated by PLP in light fractions) is beginning to develop at this early state of myelination and is thus underrepresented in the light fraction. Claudin11/OSP, which delineates the non-compact domain, is used as an additional marker. Syntaxin 6 was used as a marker of Golgi and endosomal membranes.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Chase of surface biotinylated PLP, MAG and MOG through endocytosis and recycling. Primary cultured oligodendrocytes were cell-surface biotinylated at 4°C and incubated for 0, 4 and 24 hours at 37°C. (A) Streptavidin-FITC staining of permeabilized cells to visualize cell-surface localization (0 hours, arrow), endocytosis (4 hours, arrowhead), and recycling of biotinylated proteins (24 hours, asterisks; see also supplementary material Fig. S4). Within 24 hours, biotinylated proteins recycle from the cell body to the periphery where they localize to myelin-like membrane sheets (asterisks). 3D stacks of confocal planes are shown. Scale bars: 5 µm. (B) Subfractionation of primary oligodendrocytes as described in Fig. 6 directly after biotinylation (0 hours) and after a chase period of endocytosis and recycling for 24 hours at 37°C. Biotinylated proteins were precipitated from the fractions using NeutrAvidin-beads and analyzed by western blotting (a). Within 24 hours, biotinylated PLP and MOG significantly shift from heavy (H) to light fractions (L). MAG remains associated with the heavy (H) fraction. As expected, endosomal syntaxin 6 is not biotinylated and does not precipitate with NeutrAvidin (negative control). Western blots of the total fractions are also shown (b). The distribution of total PLP, MAG and MOG between the fractions does not significantly change over 24 hours at 37°C. Note that gels were run under nonreducing conditions, favoring dimeric PLP. (C) Densitometric quantification of NeutrAvidin-precipitated proteins from three independent experiments. The relative distribution of biotinylated PLP (monomeric + dimeric), MAG and MOG between the fractions is shown as a percentage. Error bars represent s.e.m.; *P<0.05; **P<0.01 (paired t-test).

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