Figure 8.

FIP3 regulates neuronal migration in the IZ through the interaction with Arf6 and Rab11. A, Immunoblot analysis showing the efficiency of FIP3 shRNAs (FIP3 KD#1 and #2) in primary cortical neurons. The numbers indicate the percentage of endogenous FIP3 expression level relative to α-tubulin. Note that the expression of FIP3 KD#1 and #2 decreased endogenous FIP3 by 37.0 and 65.6%, respectively. B, C, E, Representative images of coronal sections of E17.5 (Band E) and P0 (C) cerebral cortices electroporated with Control, FIP3 knockdown (B and C), or FIP3 KD#2 and FIP3^WT, FIP3^ΔABD, or FIP3^ΔRBD (E) in combination with pCAGGS-EGFP at E14.5. The bottom graphs show the quantification of the distribution of EGFP-positive neurons in each layer. n in the graph indicates the number of embryos examined. D, Immunoprecipitation. HeLa cells were transfected with the indicated combinations of plasmids and subjected to immunoprecipitation with anti-FLAG affinity gel. Immunoprecipitates and total lysates were subjected to an immunoblot analysis with anti-HA or anti-FLAG antibodies. Note the lack of the ability of FIP3^ΔABD and FIP3^ΔRBD to interact with Arf6 and Rab11, respectively. WB, Western blot. F, Comparison of the percentage of EGFP-positive cells transfected with the indicated plasmids in the CP at E17.5. Note the ability of FIP3^WT but not FIP3^ΔABD or FIP3^ΔRBD to partially rescue the disturbed cortical layer formation caused by the knockdown of FIP3. G, Representative time-lapse images of transfected neurons in the IZ. Embryos were electroporated with the indicated shRNAs, pCAGGS-Cre recombinase, and pCAGGS-Floxp carrying EGFP, EGFP-NLS, and EGFP-Fyn at E14.5, and subjected to time-lapse observations at E17.5. Arrowheads indicate transfected neurons. H, Quantification of the migration speed of transfected neurons in the IZ (n = 40 cells from 3 embryos). Note the decrease in the migration speed of FIP3 KD#2-transfected neurons compared with that of control neurons. I, Tracking analysis of migration of transfected neurons in the IZ. The graphs show the trajectory of migrating neurons for 10–13 h (n = 40 cells from 3 embryos). Data were presented as mean ± SEM (B, C, E, F) and mean ± SD (H), and statistically analyzed using one-way ANOVA followed by post hoc Tukey-Kramer’s test in B (vs control) and E (vs FIP3 KD#2 in B; *P < 0.05, ** P < 0.01), and unpaired t test in C and H(vs control; *P < 0.05, ** P < 0.005, ***P < 0.0005). The total numbers of examined animals were indicated in the graphs in B, C,and E. Scale bars: B, C, E, 200 μm; F, 50 μm.