Extended Data Fig. 6: mDia1 binds to the SPIN90-Arp2/3 complex.

a, mDia1 is dose-dependently eluted with Arp2/3 bound to GST-SPIN90 decorated beads. Left: Anti-His and Anti-ArpC2 western blots of GST pull down assay. GST beads were incubated with 12 μM GST-SPIN90 and with the indicated amounts of Arp2/3. The beads were washed and incubated with 750 nM mDia1. Right: quantification of pulled-down mDia1, normalized to GST-SPIN90 without Arp2/3 (mean ± SD, n = 3 independent experiments). Black dots show the data points. For each concentration, the red dot shows the mean and the red bars the standard deviation. Uncropped Western blot can be found in Source data figure ED6. b, Left: schematic diagram of a control experiment (related to Fig. 6C-D). Control experiments consist in observing the elongation of new, bare barbed ends growing in a microfluidics chamber with a SPIN90-decorated surface exposed to Arp2/3 and mDia1. This is achieved by first following the sequence shown in Fig. 6C, and then photo-severing the filaments with a strong laser illumination (see Methods). We then observed the regrowth of filaments from SPIN90-Arp2/3. The photo-severing ensured that the newly elongating barbed ends were initially without mDia1. The distribution of slow (light blue), slow then fast (blue), and fast (dark blue) filaments was compared to experiments like the ones presented in Fig. 6C-D (bar charts, on the right, n=number of filaments). “Complex” denotes the experiments performed as in Fig. 6C-D (with an additional exposure to light, to have the same conditions as in the control, see Methods) and “control” denotes the control experiments. Comparison using one-tailed Fisher’s exact test: p = 0.31 for the slow-then-fast population, and p = 0.0013 for the fast population. n is the number of filaments randomly picked and investigated in an independent experiment. The experiment was repeated twice independently with similar results. Statistical source data can be found at Source data figure ED6.

Source data