Figure 8. In Fig4^-/- OLs, MAG accumulates in large perinuclear vacuoles.

Confocal images of live OLs acutely labeled with bath applied anti-MAG-Alexa488 (green) and LysoTracker Deep Red. (A-A’’) Fig4 control (Fig4^+/+ or Fig4^+/-) OLs incubated with anti-MAG-Alexa488 and LysoTracker, single channel and merged images are shown. (B-B”) Fig4^-/- OLs incubated with anti-MAG-Alexa488 and Lysotracker shows accumulation of MAG in large perinuclear vacuoles (arrows), single channel and merged images are shown. Of note, large perinucler MAG⁺ vacuoles do not stain with LysoTracker. (C) Scatter plot depicting the volume of anti-MAG-Alexa488⁺ particles in live Fig4 control and Fig4^-/- OLs. Each dot represents an individual vesicle (n = 4 experiments, 9 cells per genotype). Mean volumes ± SEM are shown. (D-D”) Wildtype OLs were incubated with anti-MAG-Alexa488 and LysoTracker and then acutely treated with the PIKfyve inhibitor apilimod. MAG accumulates in large perinuclear vacuoles, the majority of which does not stain with LysoTracker (n = 4 for Fig4 controls and n = 4 for Fig4^-/-cultures). For apilimod treatment, n = 3 independent cultures. Maximum projection confocal z-stack images are shown, scale bar = 10 μm. Further characterization of enlarged perinuclear vacuoles in Fig4^-/- OL cultures, specificity control for the anti-MAG-Alexa488 antibody and distinct trafficking routes of MAG and MOG are shown in Figure 8—figure supplement 1–3 and 4.

DOI: http://dx.doi.org/10.7554/eLife.13023.021

Figure 8—figure supplement 1. Fig4^-/-OLs show enlarged perinuclear vacuoles that stain positive for LAMP1.

Confocal images of (A-A”) Fig4 control (Fig4^+/+ or Fig4^+/-) and (B-B”) Fig4^-/-OPCs cultured for two days in the presence of PDGF, fixed and double-stained with anti-LAMP1 and anti-PDGFRα antibodies. TO-PRO-3 dye was added to stain nuclei. Few OPCs (<20%) in Fig4^-/- cultures showed enlarged LAMP1⁺ vesicles (white arrows). (C-D’’) Confocal images of (C-C”) Fig4 control and (D-D”) Fig4^-/- OLs after 4 days in T3 containing differentiation medium. Cultures were fixed and double-stained with anti-LAMP1 and anti-MAG antibodies. TO-PRO-3 dye was added to stain nuclei. In Fig4^-/- cultures, the majority of OLs (>65%) showed multiple large perinuclear vacuoles that were double-positive for LAMP1and MAG (white arrows). Observations were made in 4 independent experiments per culture condition. Scale bar, A-D” = 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.13023.022

Figure 8—figure supplement 2. In Fig4^-/- OLs, PM derived MAG is transported to enlarged vesicles in the LE/Lys compartment.

Representative confocal images of (A) Fig4 control OLs and (A’) Fig4^-/- OLs transfected with a Rab7-YFP expression construct. Large perinuclear Rab7-YFP⁺ vesicular structures are observed in Fig4^-/- OLs (arrows). Scale bar = 20 μm. Confocal images of (B-B”) Fig4 control (Fig4^+/+ or Fig4^+/-) and (C-C”) Fig4^-/- OL cultures transfected with a LAMP1-mCherry expression construct and incubated in bath applied anti-MAG-Alexa488 antibody. (B”) In Fig4 control cultures MAG is localized to LAMP1⁺ vesicles with a diameter of less than 1 µm. (C”) In Fig4^-/-cultures, MAG labeling is frequently observed in enlarged perinuclear and LAMP1⁺ vesicles (arrows). Scale bar = 20 μm.

DOI: http://dx.doi.org/10.7554/eLife.13023.023

Figure 8—figure supplement 3. Specificity control for anti-MAG-Alexa488 antibody.

Live-cell imaging of primary OLs prepared from Mag^+/+ and Mag^-/-pups following bath application of anti-MAG-Alexa488 (green) and LysoTracker Deep Red. Representative confocal Z-stack images are shown. (A-A”) Anti-MAG-488 labeling of intracellular vesicles is robust in Mag^+/+ OLs. (B-B”) No signal is detected in parallel processed Mag^-/- OLs. Independent of MAG genotype, prominent LysoTracker staining is observed. (C-D’’) To rule out the possibility that large vacuoles are non-specifically labeled by anti-MAG-Alexa488, Mag^+/+ and Mag^-/-OL cultures were treated with the PIKfyve kinase inhibitor apilimod. Apilimod leads to accumulation of enlarged perinuclear vacuoles in Mag^+/+and Mag^-/-cultures. (C-C’’) In Mag^+/+ cultures, vacuoles are strongly labeled with anti-MAG-Alexa488. (D-D’’) In Mag^-/- cultures, no labeling with anti-MAG-Alexa488 was observed. Scale bar in A-D” = 7.5 µm.

DOI: http://dx.doi.org/10.7554/eLife.13023.024

Figure 8—figure supplement 4. Live imaging of primary OLs reveals distinct trafficking routes for PM-derived MAG and MOG.

Confocal images of (A-A”) Fig4 control and (B-B”) Fig4^-/- OLs simultaneously incubated with anti-MAG-Alexa488 and anti-MOG-Alexa555 antibodies. Independent of Fig4 genotype, there is little overlap among MAG⁺ (green) and MOG⁺ (red) structures. In Fig4^-/- OLs, enlarged MAG⁺ vesicular structures (arrows) are MOG^-. Scale bar A-B’ = 7.5 µm.

DOI: http://dx.doi.org/10.7554/eLife.13023.025