Fig. 1: Leading process morphology and growth cone dynamics in MGE cells expressing PAK3 mutants. | Molecular Psychiatry

Fig. 1: Leading process morphology and growth cone dynamics in MGE cells expressing PAK3 mutants.

From: PAK3 activation promotes the tangential to radial migration switch of cortical interneurons by increasing leading process dynamics and disrupting cell polarity

Fig. 1

A Scheme of the electroporated murine PAK3 constructs (see Results). In vitro kinase assay shows that the PAK3-wt protein is activated by active GTPases, whereas the PAK3-ca protein displays high constitutive kinase activity and the PAK3-kd protein is totally devoid of activity. First row, auto-phosphorylation of mutants; second row, purified PAK3 mutants; third row, expression of GTPases in transfected cells. B To prepare co-cultures, MGE explants were dissected out of telencephalic vesicles at E13.5, electroporated and placed on a substrate of E13.5 wild type dissociated cortical cells. MGE cells migrated centrifugally away from MGE explants on the substrate of cortical cells. Leading process morphology was analyzed in fixed co-cultures (C–E). Dynamic transformations of growth cones were analyzed by time-lapse video-microscopy in living co-cultures (F, G). C Pictures show MGE cells expressing control eGFP (C1, black dot), eGFP-PAK3-ca (C2, red dot) and eGFP-PAK3-kd (C3, blue dot) constructs at the migration front. Scale bar, 20 μm. D Graph of the length of the longest neurite in large samples of electroporated cells (numbers below graphs). Lengths were normalized to the mean value in the control sample (eGFP expressing cells). Statistical significance was tested by the Kruskal–Wallis test (p < 0.0001) and Dunn’s post-hoc tests. E Histogram showing the percentage of MGE cells exhibiting a leading process without (black), with 1 (dark grey), 2 (medium grey), 3 (light grey), 4 or more (white) bifurcations (chi2 tests). F Histogram showing the mean surface (pixel number) of growth cones at their maximum size on movies. Values were normalized with regards to the mean growth cone size in MGE cells expressing eGFP (One Way ANOVA, p < 0.0001, and Bonferroni’s post-hoc tests for pair comparisons). G Histograms of the mean frequency of leading growth cone splitting (G1), collapse (G2), transient collapse (G3) and growth cone neoformation leading to a novel branch (G4) in MGE cells expressing eGFP (19 cells), eGFP-PAK3-ca (18 cells), and eGFP-PAK3-kd (12 cells) constructs analyzed for more than 2 h. In G1 and G2, statistical significance is assessed by the Kruskal-Wallis test (p = 0.0004 in H1, P < 0.0001 in G2) and Dunn’s post-hoc tests. In G3 and G4, statistical significance is assessed by the Poisson-ANOVA model, and post-hoc likehood ratio test, ***p = 0.0007, **p = 0.0031 in G3, and *p = 0.036, p = 0.0116 in G4. See also Supplementary Fig. S1A and Supplementary movie S1A.

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