Figure 5. ADF/Cofilin1 activity is required for F-actin disassembly in oligodendrocyte.
(A) Oligodendrocytes were cultured from ADF/Cofilin1 KO (AC DKO) and littermate control mice and stained for F-actin using rhodamine-phalloidin and with antibodies against galactosylceramide (O1) to visualize the cell surface (DIV6). Quantification of F-actin fluorescence intensity (mean arbitrary units, AU) and cell surface projection area (n=26-52 cells from three experiments, bars show mean ± SEM, **p < 0.01, ***p < 0.001, t-test). Scale bars, 10 μm. (B) Oligodendrocytes were cultured from controls (ADF KO) and ADF/Cofilin1 KO (AC DKO) mice and F- to G- actin fluorescence intensity ratios were quantified from stainings with DNaseI to visualize G- and phalloidin-rhodamine to label F-actin (n=17-40 cells from three experiments, bars show mean ± SEM, **p < 0.01, ***p < 0.001, t-test). (C) Localization of F-actin and MBP in spinal cord sections (400 nm thick) of control and ADF/Cofilin1 DKO mice at P12. Percentage of myelinated axons showing F-actin labeling within sheaths using rhodamine-phalloidin. Bars show mean ± SEM (n=3, 874-1266 myelinated axons per animal, ***p < 0.001, t-test). Scale bars, 5 μm. (D) Sections of P17 mouse spinal cords from control (ADF KO) and ADF/Cofilin1 DKO (AC DKO) mice were stained to visualize G- and F-actin levels. Shown is also the ratio of F- to G-actin ratio image in pseudocolours to indicate the high intensitiy pixels. Quantification of F- to G-actin fluorescence intensity ratios in the grey (area outside of the white line) and white matter (indicated by a white line) (n>10 images from four animals, bars show mean ± SEM, *p < 0.01, t-test). Scale bars, 10 μm.
See also Figures S4 and S5