Fig. 6: JMY’s first WH2 domain interacts with actin most strongly, is key in JMY-mediated dendritic arbor formation, and is controlled in a CaM/Ca²⁺-dependent manner.

A Fluorescence microscopy image of specific recruitment of purified, Alexa Fluor™ 568-labeled actin by purified, individual GST-JMY WH2 domains immobilized to beads (visualized live 30 min after incubation start). GST-Cobl WH2#2 (strong G-actin-binding WH2 domain) was used as positive control. Bars, 50 μm. B Measurements of fluorescence intensities during the polymerization of monomeric pyrene actin in absence and presence of individual WH2 domains of JMY. Cobl WH2#2 was used for comparison. Representative fluorescence intensity curves out of 3 assays conducted. Note that all three WH2 domains of JMY inhibited spontaneous actin polymerization, and that JMY WH2#3 had the lowest G-actin quenching effect when all three WH2 domains of JMY were compared to each other. C, D Immunoblot analyses of coimmunoprecipitation analyses of lysates of L929 cells expressing GFP or GFP-fusion proteins of JMY CT, JMY WH2#1, JMY WH2#2, and JMY WH2#3 (C; arrows mark specific coimmunoprecipitations of endogenous actin) and quantitative analyses thereof (D). The quantitative data are expressed as coimmunoprecipitated actin per immunoprecipitated GFP fusion protein normalized to the ratio obtained with GFP-JMY CT in the respective assay (in percent). Note that only GFP-JMY CT and GFP-JMY WH2#1 specifically coimmunoprecipitated endogenous actin. E Representative MIPs of anti-MAP2-stained primary hippocampal neurons transfected at DIV4 as indicated and fixed 30 h later. Transfected cells are marked by asterisks. Bars, 30 μm. F-H Quantitative analyses of dendritic morphology of primary hippocampal neurons transfected as indicated based on anti-MAP2 staining. The neurons showed JMY RNAi-mediated decreases in dendritic branch points (F), dendritic terminal points (G), and total dendritic length (H), which were rescued by reexpression of JMY* but not by JMY*^WH2#1mut. I In vitro reconstitution of JMY WH2#1/CaM/actin complexes with purified proteins (GST-JMY WH2#1 immobilized) demonstrating the reciprocal Ca²⁺ dependence of the actin and of the CaM binding of JMY WH2#1. J Quantitative analyses of the JMY WH2#1/CaM/actin complex formation showing a highly significant decrease of actin binding and an increase of CaM binding in the presence of Ca²⁺. K, L Immunoblotting of coimmunoprecipitates of endogenous actin with GFP-JMY fragments expressed in L929 cells (K) and quantitative analyses thereof (L). The actin CoIP/GFP IP ratios expressed in percent difference from the respective Ca²⁺ free condition demonstrate a strong reduction of the actin binding of GFP-JMY CT as well as of GFP-JMY WH2#1 upon addition of 2 µM Ca²⁺ (+) compared to the Ca²⁺ free (EGTA containing) condition (−). M Quantitative analyses of coimmunoprecipitation experiments addressing the reversibility of the modulation of JMY’s actin binding by 2 µM Ca²⁺ by subsequent chelation of the Ca²⁺ with EGTA (GFP-JMY CT was used for analyses). Data, mean ± SEM shown as bar/dot plots. D GFP-JMY CT, n = 9; GFP-JMY WH2#1, n = 9; GFP-JMY, WH2#2 n = 7; GFP-JMY WH2#3, n = 7 independent sets of coimmunoprecipitations. F–H Scr. RNAi, RNAi#1, RNAi#1/JMY* and JMY*^WH2#1mut n = 30 neurons from two independent assays (data partially repeated from Fig. 5). J n = 3 sets of comparisons. L GFP-JMY CT, n = 5 independent coimmunoprecipitation assays each; GFP-JMY WH2#1 n = 4 independent coimmunoprecipitation assays each. M GFP-JMY CT (EGTA), n = 6; GFP-JMY CT (Ca²⁺), n = 6; GFP-JMY CT (Ca²⁺ and subsequently EGTA), n = 4 independent quantitative coimmunoprecipitation assays. Numerical data and images of uncropped and unedited blots are provided in Supplementary Data 1 and 2, respectively. Statistical significances, Kruskal–Wallis test/Dunn′s multiple comparison test (D), One-way ANOVA/Tukey’s post-test (F–H, M), unpaired student’s t-test (J), and two separate Welch′s t-tests (K), respectively. *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.