Figure 7

Drebrin phospho-dead and phospho-mimetic mutants overexpression affects F-actin dynamics and growth cone formation. (a) Expression of Drebrin phospho-dead mutant (DrebrinS142A) decreased F-actin dynamics and Drebrin phospho-mimetic mutant (DrebrinS142D) increased F-actin dynamics in growth cones (kymographs from white lines). (b) Quantification of F-actin treadmilling in growth cones of cells expressing Drebrin, DrebrinS142A and DrebrinS142D. Drebrin = 1.579 ± 0.1030 μm/min; n = 12 cells from at least three different cultures; DrebrinS142A = 1.090 ± 0.0659 μm/min; n = 10 cells from at least three different cultures; DrebrinS142D = 2.08 ± 0.0672 μm/min; n = 10 cells p < 0.0001 by two-way ANOVA, post hoc Tukey’s test ***p < 0.001, ****p < 0.0001; Mean ± SEM. (c) Quantification of total neurites, neurites with growth cones (gc) and growth cones with enriched Drebrin from stage 2 and 3 cells transfected with Drebrin-YFP, DrebrinS142A-YFP, and DrebrinS142D-YFP were shown. Separate statistical comparisons were made to analyze the differences in the number of neurites with growth cone (black + grey bars) and the number of growth cones with enriched Drebrin (black bars) among the groups. Number of Drebrin-enriched growth cones per cell: Drebrin WT = 1.469 ± 0.1626; DrebrinS142D = 2.300 ± 0.4236; DrebrinS142A = 0.6216 ± 0.1362; Drebrin WT n = 49, DrebrinS142D n = 20, DrebrinS142A n = 37 cells from at least three different cultures, p < 0.0001 by one-way ANOVA, post hoc Dunnett’s test, *p < 0.05, **p < 0.01. Total number of neurite (white + black + grey bars): Drebrin WT = 6.551 ± 0.2124 VS DrebrinS142A = 7.486 ± 0.5164 or DrebrinS142D = 5.950 ± 0.4070, Drebrin WT n = 49, DrebrinS142A n = 37, DrebrinS142D n = 20 cells from at least three different cultures, p = 0.0366 by one-way ANOVA, post hoc Dunnett’s test. Number of neurites with growth cone per cell: Drebrin WT = 3.122 ± 0.1812 VS DrebrinS142A = 2.216 ± 0.2424, Drebrin WT n = 49, DrebrinS142A n = 37 cells from at least three different cultures, p = 0.0004 by one-way ANOVA, post hoc Dunnett’s test, **p < 0.01. Mean ± SEM. (d–e) Confocal images of rat hippocampal cells transfected with Drebrin WT-YFP (d), DrebrinS142A-YFP (e), and DrebrinS142D-YFP (f) were treated with 5 µM Cytochalasin D at DIV2 and fixed with 4% PFA at DIV3 and stained with α-tubulin antibody (shown in red). (g) Quantification of neurite length (µm) after Cytochalasin D treatment. Untransfected control = 42.09 ± 2.813, Drebrin WT = 37.18 ± 3.350, DrebrinS142A = 37.55 ± 2.739, DrebrinS142D = 39.57 ± 2.696, Untransfected control n = 20, Drebrin WT n = 15, DrebrinS142A n = 15, Drebrin S142D n = 20 cells, P = 0.6466 by one-way ANOVA, post hoc Dunnett’s test. Mean ± SEM. Scale bar: 10 μm (a,d,e and f).