Fig. 5: CIP4-FDR phase separates and promotes actin polymerisation.

a (Top) A charge plot of CIP4-FDR. HR1 is flanked by net positive (marked in blue) and negative (marked in red) IDRs. (Bottom) A schematic illustration of phase separation of CIP4-FDR driven by charge block interaction. b In vitro phase separation of FDRs from CIP4 and FBP17 and IDR from Synd2. The turbidity of the solution was measured based on the optical density at 600 nm and plotted against the protein concentration. The saturation concentration (C_sat) was obtained via curve fitting (Methods) and is indicated. A representative microscopic image of the CIP4-FDR droplet is also presented. Similar results were obtained from three independent experiments. For fluorescence imaging, the protein was pre-labelled with ATTO488. The scale bar is 50 μm. c The summary of in vitro phase separation assay. The C_sat. was obtained from the turbidity assay and is summarised. P values were calculated using the two-tailed Student’s t test, ***P < 0.001, α = 0.05. The exact P values are provided in Source Data. Error bars represent the standard deviation from three independent experiments. All data points are shown. n.d. not determined. d FRAP analysis of a protein droplet formed by CIP4-FDR. The protein droplet of ATTO488-labelled CIP4-FDR was prepared as indicated in (b). Half of the target droplet (dotted square) was bleached via laser radiation, and time-lapse imaging was continued every 10 s. Some representative images are presented. The average fluorescence intensity of the bleached region was quantified and plotted against time as a value relative to that of the pre-bleached signal. Data are presented as mean ± standard deviation from three independent measurements. e (top) A schematic illustration of CIP4-FDR and its deletion mutants. (bottom) The turbidity assay was performed and plotted as described in (b). f (Left) Time-lapse fluorescence images of EGFP-fused CLTB and mCherry-fused CIP4(ΔIDR2/SH3) expressed in Cos7 cells. The image size is 1.0 × 1.0 µm². Time 0 was when the clathrin signal disappeared. Additional cases are indicated in Supplementary Fig. 6g. (Right) Assembly rate of CIP4 at CCPs was plotted (N = 20 pits examined over three independent experiments, for each condition). The median and lower and upper quartiles are indicated in the box. The minimum and maximum values are indicated with upper and lower whiskers, respectively. P values were calculated using the two-tailed Student’s t test, ****P < 0.0001; ns: not statistically significant, α = 0.05. The exact P values are provided in Source Data. g The self-assembly of CIP4 is not affected by Cdc42. The relative turbidity of CIP4 (FDR and FL) was measured by the turbidity assays and summarised. Data are presented as mean ± standard deviation from three independent experiments. All data points are shown. h Representative microscopic images of the co-separation of CIP4-FL or CIP4-FDR and N-WASP. Similar results were obtained from three independent experiments. For fluorescence imaging, the protein was pre-labelled with ATTO488 or ATTO610. Scale bar, 50 μm. i (Top) Schematic illustration of actin polymerisation in a protein droplet of CIP4-FDR. After forming a droplet of ATTO488-labelled CIP4-FDR, rhodamine-labelled G-actin was incubated with ATP, Mg²⁺, and K⁺. (Bottom) Time-lapse fluorescence observation of CIP4-FDR (ATTO488-labelled) and G-actin (rhodamine-labelled). Scale bar, 100 μm. j (Left) A mixture of rhodamine-labelled and non-labelled G-actin (1:9) was loaded with or without ATP and Mg²⁺ and incubated with a pre-formed CIP4-FDR droplet. Representative fluorescence images are presented here. Enlarged images in the presence of ATP are shown below. Scale bar, 10 μm. (Right) Fluorescence intensity of F-actin in the droplet was quantified and plotted (N = 60 in each condition).