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First published online 14 February 2006
doi: 10.1242/jcs.02785

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A role for Sec8 in oligodendrocyte morphological differentiation

¹ Program of Molecular Biology and Biochemistry, University of Connecticut Medical School, Farmington, CT 06030, USA
² Department of Neuroscience, University of Connecticut Medical School, Farmington, CT 06030, USA
³ Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow, G12 8QQ, UK

View larger version (62K): [in a new window]   Fig. 1. The exocyst components Sec8 and Sec6 are present in OLs and myelin. (A) OL lineage cells, myelin, brain homogenate and astrocytes were analyzed by immunoblot (10 µg protein/lane). The levels of Sec8 and Sec6 were normalized to actin and quantified for n=3 independent OL cultures. (B-E) Sec8 (green) colocalization with (B) TGN38 (red) and (C) LAMP2 (red) in OL processes was assessed by confocal microscopy and ratiometric particle analysis for (D) 434 and (E) 560 randomly chosen puncta in eight representative OLs, two independent cultures per condition. (B) The arrows indicate colocalization between TGN38 and Sec8, shown at a higher magnification in the inset. (F) Sec8 (green) is detectable along MBP-positive (red) myelinated axons in mouse spinal cords sectioned longitudinally (left panel) or transversally (right panel). No immunofluorescence was detected when cells and/or sections were incubated with pre-immune IgGs and secondary Abs (data not shown). EP, early OL progenitors; LP, late OL progenitors; OL, mature OLs; BH, whole-brain homogenate; My, myelin; As, astrocytes. Bars, 10 µm (B,C), 2 µm (F).

View larger version (57K): [in a new window]   Fig. 2. Sec8 overexpression favors OL morphological differentiation. (A-D) OL progenitors were infected overnight with adenoviruses encoding (A) control EGFP (EGFP-AV) or (B-D) FLAG-tagged Sec8 (Sec8-AV), allowed to differentiate for 3 days, fixed, labeled with anti-MBP- (red) and anti-FLAG- (green) Abs and analyzed by epifluorescence microscopy. OLs were assigned to three morphological categories: (B) low, (C) medium or (D) high complexity. Cell bodies were overexposed in order to observe protein localization in the processes. Bars, 10 µm; px, pixels. (E) Mature MBP-positive cells were plotted as percentage of the total infected cells. (F) The number of OLs in each morphological category is shown as a percentage of the total MBP-positive OLs (500 cells/condition, n=6 from three independent cultures). (G) The areas of 100 randomly chosen MBP-positive cells per condition were measured and plotted. Only infected cells were analyzed. (E-G) White bars, control EGFP-AV; black bars, Sec8-AV infected.

View larger version (18K): [in a new window]   Fig. 3. Sec8 overexpression increases the levels of a subset of myelin proteins. OL progenitors were infected with Sec8-AV and either (A) control EGFP-AV or (C) control null-AV and allowed to differentiate for 3 days in defined medium. Cell lysates were analyzed by immunoblot for (A) Sec8, Sec6, OSP/Claudin11, CASK (10 µg protein/lane); MAG, CNP, MBP, actin (2 µg protein/lane) and (B), and data are presented as percentage of control levels. (C) Cell lysates were analyzed for Sec8, MBP, CNP and actin (10 µg protein/lane). n=5 from three independent cultures, * indicates statistically significant differences.

View larger version (44K): [in a new window]   Fig. 4. siRNA-mediated interference with Sec8 expression decreases OL morphological differentiation. OL progenitors were transfected with (A) fluorescently tagged siRNAs (siGloCipB, red) and (B-E) Sec8 or control siRNAs. Arrows in A indicate a siGlo-positive perinuclear punctum. Cells were allowed to differentiate for 2 days and analyzed by (A,C) fluorescence microscopy for MBP and Hoechst or (B) immunoblot for Sec8, Sec6, MBP and actin (n=8 from four independent cultures). (D) The number of mature MBP-positive cells was plotted as a percentage of the total Hoechst-labeled nuclei (5000 cells/condition, n=11, four independent cultures). (E) Morphological complexity of CNP-positive OLs was analyzed for 900 cells/condition (n=6, two independent cultures). Bars, 10 µm.

View larger version (83K): [in a new window]   Fig. 5. Ab perturbation of Sec8 function significantly influences OL morphology. Mature OLs in culture were microinjected with dextran (red) and either anti-Sec8 Ab or IgG; 60 minutes after injection, cells were fixed and labeled with anti-MBP Ab (green). Cells were analyzed into three groups: (a) group 1: cells with fragmented processes and/or membranes; (b) group 2: cells with small areas and short processes (low and medium morphological complexity) and (c) group 3: cells with high complexity. Cells in each group are shown as percentage of the total dextran-positive injected cells (173 cells injected with anti-Sec8 Ab; 112 cells injected with IgG; n=3 independent experiments; P<0.003). Bars, 10 µm.

View larger version (82K): [in a new window]   Fig. 6. Sec8 associates with CASK and OSP/Claudin11 in OLs. (A) Sec8, CASK and OSP/Claudin11 coimmunoprecipitate. Mature OL lysates were incubated with anti-Sec8 Ab (2E12), anti-CASK Ab or pre-immune IgG and immunoprecipitates were analyzed by immunoblot. IP, immunoprecipitate; S, 10% of the supernatant fraction. (B) OSP/Claudin11, Sec8 and CASK cofractionate and float to low densities on sucrose gradients. The 1% Triton X-100 insoluble pellet fractions were isolated from mature OL lysates at 4°C, floated on sucrose-step gradients and analyzed by immunoblot; S, 15% of the soluble fraction. (C-E) Colocalization of (C) CASK (green) and OSP/Claudin11 (red), (D) Sec8 (green) and OSP/Claudin11 (red) or (E) Sec8 (green) and MOG (red) in OL processes was analyzed by confocal microscopy and ratiometric particle analysis for 680 randomly chosen puncta in 5-7 representative cells, three independent cultures. The Abs used for colocalization analysis were against the intracellular C-terminal regions of OSP/Claudin11 and MOG. Bars, 10 µm. Arrows indicate fluorescent puncta positive for OSP and CASK (C), positive for OSP and Sec8 (D), or positive for MOG, but not for Sec8 (E).

View larger version (70K): [in a new window]   Fig. 7. CASK is present in mobile vesicle-like structures in OLs. (A,B) OL progenitors in culture were transfected with a CASK-EGFP fusion protein and OLs were analyzed by confocal microscopy after 3 days. Images were converted to grayscale mode and colors inverted; CASK-EGFP is shown in black. (B) Time-lapse analysis of CASK-EGFP in a representative OL process segment shows that the protein is present in mobile puncta (arrows). Images were collected every 8 seconds. (C) OLs in culture labeled with anti-CASK Ab (green) and anti-dynein Ab (red) were analyzed by confocal microscopy and colocalization in OL processes was quantified for 280 puncta in eight representative cells from two independent cultures. Arrows indicate a fluorescent punctum positive for CASK and dynein. Bars, 10 µm (A,C), 2 µm (B).

View larger version (86K): [in a new window]   Fig. 8. CASK is detectable in MBP-positive OLs and myelinated axons in cocultures and in vivo. (A) OL-neuronal cocultures were immunolabeled with anti-MBP Ab (red) and anti-CASK Ab (green), and analyzed by confocal microscopy followed by the 3D reconstruction of Z-stacks of images. Arrows indicate MBP-positive OLs and arrowheads indicate myelinated fibers. (B,C) CASK (green) is detectable in (B) cells with a chain-like pattern, suggestive of intrafascicular OLs (rat brain) and (C) MBP-positive myelinated fibers in longitudinal or cross-sections (mouse spinal cord). The arrow in (C) indicates a CASK-positive cluster localized in an MBP-positive myelinated fiber in cross-section. Bars, 10 µm (A-C), 5 µm (C).

View larger version (18K): [in a new window]   Fig. 9. Model. Sec8 associates with the CASK/Mint1 complex and mediates the transport of motor-driven vesicles carrying lipid- and protein-cargoes (e.g. OSP/Claudin11) along microtubules to the OL and myelin membranes.