Fig. 5: JMY’s functions in dendritogenesis rely on actin-binding and on a CaM binding region comprising the first WH2 domain of JMY.

Quantitative analyses of dendritic branch points (A), dendritic terminal points (B), and total dendritic length (C) of anti-MAP2-immunostained primary hippocampal neurons transfected at DIV4 with either scr. RNAi, JMY RNAi#1, or with JMY RNAi#1 together with RNAi insensitive JMY* and JMY*^WH2#1-3mut, respectively, and fixed 30 h after transfection. Note that JMY*^WH2#1-3mut failed to rescue any of the JMY loss-of-function phenotypes. D MIP of hippocampal neurons transfected and fixed under the same conditions as for the morphometric analyses (A–C) with additional anti-JMY immunostaining and DAPI staining showing that JMY*^WH2#1-3mut, albeit not able to rescue, was successfully expressed and available in soma and dendrites. E Immunoblot of GFP-JMY deletion mutants expressed in L929 cells coprecipitated with a CaM matrix. GFP-JMY CT and GFP-JMY WH2#1 were coprecipitated with CaM matrix under 2 µM Ca²⁺ (+), but not under Ca²⁺ free (−) conditions. The anti-GFP signal of the supernatants confirmed the expression and integrity of GFP and GFP-fusion proteins in the assay. F Coprecipitation of purified GST-JMY WH2#1 but not GST with immobilized CaM demonstrating that the Ca²⁺-dependent interaction of JMY with CaM is direct. G Schematic representation of JMY and of a mutant lacking the CaM binding region. Quantitative analyses of dendritic branch points (H), dendritic terminal points (I), and total dendritic length (J) of anti-MAP2-immunostained primary hippocampal neurons transfected at DIV4 and fixed 30 h thereafter. Note that JMY*ΔCaM failed to rescue the impairments in dendritic arborization caused by JMY RNAi. K MIP of hippocampal neurons transfected and fixed under the same conditions as for the morphometric analyses (H–J) with additional anti-JMY immunostaining and DAPI staining showing that JMY*ΔCaM, albeit not able to rescue, was successfully expressed and available in soma and dendrites. Bars, 30 µm. Data, mean ± SEM shown as bar/dot plots. A–C Scr. RNAi, RNAi#1, RNAi#1/JMY* and JMY*^WH2#1-3mut n = 30 neurons from 2 independent assays. H–J Scr. RNAi, RNAi#1, RNAi#1/JMY*, and RNAi#1/JMY*^ΔCaM n = 30 neurons from two independent assays (data for the first three conditions also included in Fig. 4G–I). Numerical data and images of uncropped and unedited blots are provided in Supplementary Data 1 and 2, respectively. Statistical significances, One-way ANOVA/Tukey’s post-test (A–C, H–J). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For exact p values see figure panels. Note that ****p < 0.0001 are too small values to be reported by the software used.