Fig. 6: Oligodendrocyte VAMP2/3 is required for delivery of myelin adhesion proteins and node of Ranvier formation.
From: CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes
a Biotinylation of surface proteins on mature oligodendrocytes differentiated in culture followed by immunoprecipitation (IP) with streptavidin beads for mass spectrometry. Created with BioRender.com. b Proteomic analysis of immunoprecipitated surface proteins and interactors from control vs. iBot;Cnp-Cre oligodendrocytes. Data points exclude non-specific interactors of the streptavidin IP identified in the non-biotinylated samples. Statistical measurements were determined from two-sample T-test with a Benjamini-Hochberg false discovery rate adjustment (padj). The negative log10 padj of each specific interactor was plotted against its average log2 fold change between iBot;Cnp-Cre and control samples. The dotted curve corresponds to the threshold cutoff for a 2-fold change between samples and a permutation-based false discovery rate correction for multiple comparisons with p < 0.05. Magenta points highlight notable myelin proteins that are significantly dependent on VAMP2/3 for surface delivery. c Selected gene ontology (GO) annotations using gProfiler for VAMP2/3-dependent hits from the surface proteomic analysis (b) with a dotted line marking padj = 0.05. Adjusted p-values were determined by the gProfiler website, which uses the cumulative hypergeometric test with the g:SCS correction method. d Annotated localization of top VAMP2/3-dependent proteins in oligodendrocytes. The area of each circle scales linearly to the log2 fold depletion in iBot;Cnp-Cre oligodendrocytes. Lines connecting proteins denote reported and predicted protein interactions from the STRING database. Created with BioRender.com. e Immunohistochemistry of axonal node components Caspr (cyan) and AnkG (red) for longitudinal sections of the spinal cord harvested from control (top) and iBot;Cnp-Cre (bottom) littermates at P12. Images are representative of n = 4 biologically independent replicates quantified in g. Scale bar, 20 μm. f Representative images of node classifications. Scale bar, 2 μm. g Quantification of the number of nodes in each category over 48,000–72,000 μm2 of spinal cord longitudinal section for each of n = 4 biologically independent replicates. Square and circle symbols denote males and females, respectively. Mean number of nodes per 1000 μm2 ± SEM for control vs. iBot;Cnp-Cre, respectively: mature nodes 2.85 ± 0.52 vs. 0.28 ± 0.09; heminodes 2.03 ± 0.76 vs. 0.70 ± 0.19; clusters 3.93 ± 0.66 vs. 1.48 ± 0.50. Statistical significance was determined by one-way ANOVA with multiple comparisons correction. Source data are provided in the Source Data file.
