Figure 6: Interdependent regulation of the actin cytoskeleton and vesicle distribution in the growth cone by MICAL-1 redox activity.

(a) IP of myc-tagged MICAL-1 from HEK293 cells. Immunoblots were probed with the indicated antibodies: GFP (MICAL-3), myc (MICAL-1) and V5 (NDR1). Full-size western blots can be found in Supplementary Fig. 9. (b) Biotin–streptavidin pulldown of MICAL-3 from HEK293 cells. Immunoblots were probed with the indicated antibodies: GFP (MICAL-3, upper panel; and GFP control, lower panel) and myc (MICAL-1). IB: immunoblot; IP: immunoprecipitation; numbers indicate protein size in kDa. (c) Immunofluorescent staining for βIII-tubulin in growth cones of mouse hippocampal cultures derived from MICAL-1^+/+ or MICAL-1^−/− pups. Scale bar, 5 μm. (d) Immunofluorescent staining for Rab6 (white) together with phalloidin staining (red) in growth cones of mouse hippocampal cultures derived from MICAL-1^+/+ or MICAL-1^−/− pups transfected with the indicated constructs. FL WT; GW, enzymatically inactive MO domain. Scale bar, 5 μm. (e) Quantification of the intensity of F-actin in growth cones as in d. WT GFP, n=19; KO GFP, n=50; KO FL, n=27; KO GW, n=30 growth cones. One-way ANOVA, **P<0.01. Data are presented as means±s.e.m. (f) Immunofluorescent staining of F-actin and Rab6 following treatment with vehicle (Vh), 1 or 5 nM latrunculin B for 1 min. Scale bar, 5 μm. (g) Quantification of the intensity of F-actin in growth cones as in f. WT Vh, n=111; KO Vh, n=91; KO 1 nM, n=87; KO 5 nM, n=123 growth cones from three individual experiments. Data are normalized on WT vh. One-way ANOVA, ****P<0.0001. Data are presented as means±s.e.m. Vh, vehicle.