Figure 4: Changes in the cell surface and intracellular expression of PSA–NCAM in MICAL-1-deficient growth cones.

(a) Immunocytochemistry for PSA–NCAM on growth cones of 1 DIV DG granule cells obtained from P7 mice. Internal (red) and cell surface (green) pools of PSA–NCAM are visualized separately. Scale bar, 5 μm. (b) Fluorescent intensity of experiments as in (a) was quantified by measuring the surface and internal mean grey value for each growth cone. Surface levels were divided by internal levels per growth cone before normalization. All data were normalized to WT. +/+ 100 growth cones from 67 cells; −/− 116 growth cones from 68 cells, from three independent experiments. Unpaired t-test, *P<0.05, ***P<0.001. Data are presented as means±s.e.m. (c) Immunocytochemistry for GFP on growth cones of 2 DIV DG cultures transfected with NCAM–GFP cDNA. Scale bar, 5 μm. (d) Graph shows quantification of the intensity (mean grey value) of NCAM–GFP signals as shown in (c). +/+ 126 growth cones from 39 cells; −/− 107 growth cones from 33 cells, from three independent experiments. Unpaired t-test, **P<0.01. Data are presented as means±s.e.m. (e) Immunocytochemistry to detect GFP (NCAM), myc (MICAL-1) and endogenous Rab6b in transiently transfected 2 DIV hippocampal neurons from P0 WT mice. Boxed regions are shown at higher magnification at the bottom of each panel. White arrows indicate examples of vesicular structures containing MICAL-1–myc, NCAM–GFP and Rab6. Scale bar, 10 μm.