Fig. 5: Loss of Daam1-MIC impairs RHOA/ROCK signaling cascade. | Nature Communications

Fig. 5: Loss of Daam1-MIC impairs RHOA/ROCK signaling cascade.

From: A highly conserved neuronal microexon in DAAM1 controls actin dynamics, RHOA/ROCK signaling, and memory formation

Fig. 5

a Zoom-in of representative STORM images of actin (phalloidin-A647N) (white) overlaid with the conventional fluorescent image of PSD95 (green) of primary hippocampal cultures shown in Fig. S10a. Scale bar = 1 µm. b Quantification of the STORM localizations of F-actin in dendritic spines co-localizing with PDS95 signal, normalized per area. Each point represents a dendritic spine. Black lines connect the paired means of different biological replicates. P-values correspond to paired two-sided Mann-Whitney tests. c, d Interaction of DAAM1 with RHOA. c Co-immunoprecipitation experiment performed with anti-FLAG antibody. d Quantification of the interaction between DAAM1-ΔDAD constructs with ( + MIC) or without microexon (-MIC) and RHOA in panel (c) and S10e,f. N = 3 biological replicates (experiments). P-values from two-sided Wilcoxon rank-sum tests. e Confocal micrographs representing RhoA2G distribution (top) and RhoA activity (sensor ratio; bottom) in neurons. Boxes: example regions of interest. Scale bar: 50 μm. f Comparison of biosensor efficiency. One dot represents FRET efficiency in one neuron. WT: n = 59, KO: n = 57 neurons, N = 2 replicates. P-values from two-way ANOVA with replicate and genotype as factors. g Scatter plot showing changes in gene expression (log2 fold changes) between WT and KO cells in the control treatment (Veh) (X-axis) vs. KO control cells and KO cells treated with ROCK inhibitor (Y-axis). Each data point corresponds to a gene colored according to their “rescue” group: orange, rescued by ROCKi; red, not rescued; gray, other genes (see “Methods”). h Molecular Function GO term enrichment for genes with a rescue pattern (orange in (g)). i, j Calcium influx measurements upon ROCKi treatment. P-values from Kruskal–Wallis test. Control: n = 66 WT, and 51 KO neurons; ROCKi: n = 83 WT, and 88 KO neurons. k NOR experiment performed to test the effect of ROCKi. l Discrimination index values in the different conditions and for each genotype. P-values from two-sided Wilcoxon rank-sum tests. NOR test was performed twice, using six animals per treatment and genotype in each replicate. Replicates were separated by sex (24 animals per replicate).

Back to article page