Extended Data Fig. 5: mDia1 preferentially binds to filaments nucleated by SPIN90-Arp2/3.
From: SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin organization
a, Comparison of mDia1 binding to mother filaments versus branches. Preformed branched actin filaments (15% Alexa568 labeled) were mixed with 0.5 μM G-actin (15% Alexa488-labeled), 0.5 μM profilin, 0.4 nM mDia1, before being introduced into a passivated open chamber. The TIRF microscopy image shows Alexa568 in red and Alexa488 in green. Plot: fraction of rapidly growing barbed ends (i.e. bearing a formin) observed after 90 s, within the population of mother filaments (dark blue bar) and branches (light blue bar). Comparison with Pearson’s chi square test: p = 0.88. b, Comparison of mDia1 binding to spontaneously nucleated versus SPIN90-Arp2/3-nucleated filaments. Spontaneously nucleated actin filaments (15% Alexa568-labeled) were mixed with SPIN90-Arp2/3-nucleated actin filaments (15% Alexa488-labeled) and with 0.2 nM mDia1, 0.5 µM profilin and 0.5 µM G-actin (3% Alexa488-labeled). TIRF microscopy image: filaments growing from spontaneously assembled seeds (red arrow) or from SPIN90-Arp2/3-nucleated seeds (green arrow) are identified by the fluorescence at their pointed end. Plot: fraction of rapidly growing barbed ends observed after 300 s, within each population. Comparison with a Pearson’s chi square test: p = 0.009. Similar results were observed when repeating the experiment and inverting the fluorophores (Supplementary Figure 8B). c, Comparison of the fraction of mDia1-bearing barbed ends depending on filament nucleation history. SPIN90-Arp2/3-nucleated actin filaments (15% Alexa568 labeled) were mixed with 0.5 μM G-actin (15% Alexa488 labeled), 0.5 μM profilin, 25 nM Arp2/3, 250 nM SPIN90 and 0.2 nM mDia1. TIRF microscopy image: filaments nucleated before mixing (i.e. exposed to mDia1 after their nucleation by SPIN90-Arp2/3) are identified thanks to their Alexa568 (red) pointed end region. Plot: fraction of rapidly growing barbed ends observed after 300 s, within each population. Comparison with a Pearson’s chi square test: p = 9×10-7. (a,b,c) Error bars represent 95% confidence intervals. **p < 0.01 and ***p < 0.001, Pearson’s chi-squared test. For each population, n indicates the number of analysed barbed ends, in one experiment. The experiments (A, B) were repeated twice, and (C) three times, independently with similar results. Statistical source data can be found at Source data figure ED5.
