JCS022731 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Endocytic trafficking of endogenously expressed myelin proteins in primary cultured oligodendrocytes. (A) Cells cultured for 3-4 days were incubated on ice with primary antibodies recognizing cell surface epitopes of PLP, MAG, MOG or NG2 followed by Cy3-coupled secondary antibodies. After incubation for 1 hour at 4°C (control, top row) or at 37°C to allow endocytosis (bottom row), surface-localized antibody complexes were counterstained with Cy2-coupled antibodies. Endocytosed proteins appear red, cell surface localized proteins appear yellow. After 1 hour at 37°C, myelin proteins are efficiently endocytosed, whereas other membrane proteins such as NG2 largely remain at the plasma membrane. (B) Distinct endosomal targeting of MAG and MOG after internalization (PLP is not shown because late endosomal/lysosomal targeting has been demonstrated previously). After endocytosis for 1 hour at 37°C, surface-localized antibody complexes were counterstained with Cy5-coupled antibodies (magenta). Labeling of RE 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). Cell surface localized proteins appear magenta, endocytosed proteins appear red, colocalization with endocytic markers appears yellow. MAG colocalized with LAMP1 in LE/Lys (top left image), whereas MOG colocalized with transferrin-FITC in the RE (bottom right image). Thus, endocytic trafficking of endogenously expressed myelin proteins in primary oligodendrocytes is identical to ectopically expressed proteins in Oli-neu cells. Insets show enlarged areas. Scale bars: 5 µm.

• Supplemental Figure S2 -

  Fig. S2. Internalization of PLP, MAG and MOG is not dependent on crosslinking with secondary antibody. Oli-neu cells transiently expressing the myelin proteins PLP, MAG or MOG were incubated at 4°C with primary antibodies recognizing cell surface epitopes of PLP, MAG, or MOG and further kept for 1 hour at 4°C or 37°C to block membrane traffic or to allow endocytosis, respectively. Subsequently cells were fixed, permeabilized and immunostained with secondary Cy3-conjugated antibody (red). PLP, MAG, and MOG are internalized at 37°C (arrows) resulting in a similar subcellular pattern of distribution as observed in experiments where the cells had been exposed to secondary antibody before endocytosis: PLP is present in large vesicular structures, endosomes containing MAG scatter throughout the cell body and MOG localizes to a juxtanuclear compartment (arrows). Thus, internalization and subsequent localization of the myelin proteins occurs independent of secondary antibody incubation. Scale bars: 5 µm.

• Supplemental Figure S3 -

  Fig. S3. Fractionation of myelin isolated from adult mouse brain. Membranes were separated by discontinuous density gradient centrifugation in ‘heavy’ (H), ‘medium’ (M) and ‘light’ (L) membrane fractions, and the distribution of PLP, MAG and MOG was analyzed by western blotting. The three proteins exhibit distinct fractionation patterns, consistent with to their preferential localization to different myelin subdomains. In adult myelin, formation of compact myelin is completed and the myelin subdomains are fully differentiated. PLP is thus prominent in the light fraction, whereas MAG is prominent in heavy fractions, and MOG is spread over all three fractions.

• Supplemental Figure S4 -

  Fig. S4. Endocytosis of cell surface biotinylated PLP, MAG and MOG in primary oligodendrocytes. Primary cultured oligodendrocytes were cell-surface biotinylated at 4°C and incubated for 0 hours and 4 hours at 37°C. Subsequently, cells were fixed, permeabilized and costained using streptavidin-FITC (green) and antibodies recognizing PLP, MAG or MOG (red). Immediately after biotinylation, the streptavidin label is restricted to the cell surface (top images). After 4 hours at 37°C, biotinylated proteins have been internalized and streptavidin colocalized with myelin proteins PLP, MAG or MOG in intracellular compartments (see boxes which show enlarged areas), thus indicating endocytosis of biotinylated myelin proteins from the cell surface. Since cell surface biotinylation is highly efficient, labeling all plasma membrane proteins as well as lipids, the streptavidin staining appears overexposed compared with the areas where endocytosed biotin signal colocalizes with myelin proteins. Confocal planes were stacked using ImageProPlus 4.5 software, processing stacks in a way that only areas of colocalization appear yellow (extended depth of field function). Scale bars: 5 µm.