PSD condensates promote actin bundling via IRSp53-mediated actin interaction. (A) Fluorescence images showing the actin filament network assembled from PSD complexes in the presence of WT IRSp53. An actin binding–deficient mutation in IRSp53 abolished actin bundle assembly. A mutation that weakened the phase separation of PSD complexes also failed to promote actin bundling. Identical imaging settings were used for all groups. (B) Sedimentation-based experiments showing the distribution of PSD components in the dilute (S) and condensed phases (P) when 4× PSD was mixed with IRSp53 WT or actin binding deficient mutant. 5× PSD* refers to the mixture of PSD-95, IRSp53, GKAP, Shank3_ΔPRM, and Homer3. Each PSD component was at 5 μM. Quantifications of the sedimentation data are presented as mean ± SD. Data were obtained from three independent batches of experiments. *, P < 0.05; ****, P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test. (C) Schematic diagram showing the tethering of PSD components to the supported negatively charged lipid bilayer. (D) Representative fluorescence images showing actin filaments assembled from PSD condensates on the SLB. The actin binding–deficient mutations in IRSp53 significantly diminished actin bundling to PSD condensates on the membrane. A line plot of the Rhd-labeled actin intensities along the dashed line is presented. Identical imaging settings were used for all groups. Note that an additional panel is included for actin binding–deficient mutants of IRSp53, showing images of Rhd-actin with different contrast to the WT for better visualization. The starting concentration of the proteins in solutions (IRSp53, GKAP, Shank3, and Homer3) was 250 nM each. 10% Rhd-labeled actin was 1 μM. 10% of His8-PSD-95 and IRSp53 were labeled with the indicated fluorophores. Source data are available for this figure: SourceData F5.
Figure 5.

PSD condensates promote actin bundling via IRSp53-mediated actin interaction. (A) Fluorescence images showing the actin filament network assembled from PSD complexes in the presence of WT IRSp53. An actin binding–deficient mutation in IRSp53 abolished actin bundle assembly. A mutation that weakened the phase separation of PSD complexes also failed to promote actin bundling. Identical imaging settings were used for all groups. (B) Sedimentation-based experiments showing the distribution of PSD components in the dilute (S) and condensed phases (P) when 4× PSD was mixed with IRSp53 WT or actin binding deficient mutant. 5× PSD* refers to the mixture of PSD-95, IRSp53, GKAP, Shank3_ΔPRM, and Homer3. Each PSD component was at 5 μM. Quantifications of the sedimentation data are presented as mean ± SD. Data were obtained from three independent batches of experiments. *, P < 0.05; ****, P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test. (C) Schematic diagram showing the tethering of PSD components to the supported negatively charged lipid bilayer. (D) Representative fluorescence images showing actin filaments assembled from PSD condensates on the SLB. The actin binding–deficient mutations in IRSp53 significantly diminished actin bundling to PSD condensates on the membrane. A line plot of the Rhd-labeled actin intensities along the dashed line is presented. Identical imaging settings were used for all groups. Note that an additional panel is included for actin binding–deficient mutants of IRSp53, showing images of Rhd-actin with different contrast to the WT for better visualization. The starting concentration of the proteins in solutions (IRSp53, GKAP, Shank3, and Homer3) was 250 nM each. 10% Rhd-labeled actin was 1 μM. 10% of His8-PSD-95 and IRSp53 were labeled with the indicated fluorophores. Source data are available for this figure: SourceData F5.

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