Fig. 3: Daam1-MIC removal increases calcium flux in glutamatergic neurons.

a Schematic representation of the neuronal differentiation protocol. DIV - day in vitro. b RT-PCR assays of Daam1-MIC inclusion during neuronal differentiation in representative WT and KO cell lines. RT-PCR assays were performed once. c, d Distributions of the lengths of the longest neurites (n = 491 WT, and 517 KO neurons, N = 2) (c) and individual filopodia (N = 3) (d) of neuronal precursors (DIV0 + 4 h). P-values from two-way ANOVA tests with replicate and genotype as factors. e Representative images of the calcium imaging experiment performed using FURA-2AM in mature neuronal cells (DIV21) depolarized with 30 mM KCl isotonic solution. Scale bar: 10 μm. f Distributions of calcium influx in WT and KO cell lines at various differentiation time points, based on FURA-2AM ratio. Neurons were stimulated with a 30 mM KCl isotonic solution. DIV1: n = 217 WT, and 228 KO neurons; DIV7: n = 188 WT, and 259 KO neurons; DIV14: n = 118 WT, and 134 KO neurons; DIV21: n = 116 WT, and 146 KO neurons. P-values from one-way ANOVA Tukey’s test. g Effects of the actin polymerization inhibitor Latrunculin A (LatA) and the small molecular inhibitor of formin FH2 domains (SMIFH2) on calcium currents in differentiated glutamatergic neurons at DIV14-21. The data corresponds to three independent experiments. Top: schematic representation of the mode of action of the actin-modulating drugs. h Distributions of calcium influx in WT and KO cell lines upon different treatments as depicted in (g), based on FURA-2AM ratio. Control: n = 234 WT, and 280 KO neurons; LatA: n = 124 WT, and 144 KO neurons; SMIFH2: n = 187 WT, and 187 KO neurons.