Figure 3.

Aurora-A inhibitors severely impair neuronal migration. Migration of granular neurons after treatment of Aurora-A inhibitors was examined. a, Western blotting analysis of proteins or phosphorylated proteins. Aurora-A and NDEL1 displayed similar expression levels, whereas phosphorylated Aurora-A and NDEL1 proteins were decreased during treatment with Aurora-A inhibitors. Relative intensities of the bands of Western blotting are displayed at the bottom. Intensity was normalized with β-actin. Statistical examination was performed by unpaired Student's t test, with *p < 0.05 and **p < 0.01 (bottom panel). b, Immunocytochemistry with antibodies to Aurora-A (b) or NDEL1 (c) revealed similar expression levels, whereas phosphorylated proteins were decreased during treatment with Aurora-A inhibitors. The white dotted lines indicate the outline of granular neurons. d, Migration assays using cerebellar granule neurons. DIC images (top) and Hoechst staining images (bottom) are shown. e, The migration distance of each neuron 24 h after start of culture was binned. n is the number of neurons measured for each examination. Wild-type neurons displayed normal migration distances, whereas neurons treated with inhibitors display a shift in the distribution of bins toward the left. f, Mean length of neurites (open bar) and nuclear position (solid bar) from the edge of central aggregation. Note: Extension of neurites was not affected by inhibitors, whereas nuclear migration was significantly reduced. Statistical examination was performed to nuclear migration by an ANOVA followed by t test with correction: **p < 0.01. g, Rescue experiments of Aurora-A Inhibitor I after transfection of control GFP, GFP-Ndel1 (S251A), or GFP-Ndel1 (S251E). GFP images (top), DIC images (middle), and Hoechst staining images (bottom) are shown. Expression of GFP-Ndel1 (S251A) had no effect on migration after Aurora-A Inhibitor I treatment, whereas expression of GFP-Ndel1 (S251E) significantly improved reduced migration. h, The migration distance of each neuron 24 h after start of culture was binned. n is the number of neurons measured for each examination. Expression of GFP-Ndel1 (S251E) significantly improved migration after Aurora-A Inhibitor I treatment, which was characterized by a rightward shift of migration. i, Mean length of neurites (open bar) and nuclear position (solid bar) from the edge of central aggregation. Statistical examination was performed to nuclear migration by an ANOVA followed by t test with correction: **p < 0.01.