Multivalent interactions drive the condensed phase formation of the PSD-95/IRSp53 mixture. (A) Schematic diagrams showing the domain organization of IRSp53 and PSD-95. PDZ1 and PDZ2 tandem is referred to as PDZ12; PDZ3-SH3-GK supramodule is referred to as PSG. (B) Fluorescence images showing that IRSp53 and PSD-95 are colocalized and enriched in condensed droplets. The dashed box is selected for zoom-in view of the droplet fusion process in C. IRSp53 and PSD-95 were mixed at an equimolar ratio at 10 μM, and 2% of each protein was labeled by the indicated fluorophore. This fluorophore labeling ratio was used throughout the study unless otherwise stated. (C) Spherical droplets fuse into larger ones over time upon contact. (D) FRAP analysis of IRSp53 and PSD-95 in the condensed phase showing dynamic exchange of proteins between the condensates and the dilute solution. The concentration of each protein was 10 μM. The recovery curves of the fluorescence signals represent the averaged signals from three droplets, and data are presented as means ± SD. (E) Left, representative SDS-PAGE of sedimentation-based assays showing the amount of PSD-95 and IRSp53 recovered from the condensed phase or pellet (P) and the aqueous phase or supernatant (S) at indicated protein concentrations. Right, quantification of protein distribution in E. Results are from three independent batches of experiments and are presented as mean ± SD. (F) Left, sedimentation experiments showing the distribution of PSD-95 and IRSp53 in P and S at indicated protein concentrations. Right, quantification of protein distribution in F. Results are from three independent batches of experiments and are presented as mean ± SD. (G) Fluorescence images showing truncations of PSD-95 with weakened LLPS capability with IRSp53. The N-terminal 30 residues proceeding PDZ1 (NT in A) are not required for phase separation. (H) Fluorescence images showing truncations of IRSp53 with weakened LLPS capability with PSD-95. Replacement of the BAR domain with Myo-CC retained its phase separation with PSD-95. Source data are available for this figure: SourceData F1.
Figure 1.

Multivalent interactions drive the condensed phase formation of the PSD-95/IRSp53 mixture. (A) Schematic diagrams showing the domain organization of IRSp53 and PSD-95. PDZ1 and PDZ2 tandem is referred to as PDZ12; PDZ3-SH3-GK supramodule is referred to as PSG. (B) Fluorescence images showing that IRSp53 and PSD-95 are colocalized and enriched in condensed droplets. The dashed box is selected for zoom-in view of the droplet fusion process in C. IRSp53 and PSD-95 were mixed at an equimolar ratio at 10 μM, and 2% of each protein was labeled by the indicated fluorophore. This fluorophore labeling ratio was used throughout the study unless otherwise stated. (C) Spherical droplets fuse into larger ones over time upon contact. (D) FRAP analysis of IRSp53 and PSD-95 in the condensed phase showing dynamic exchange of proteins between the condensates and the dilute solution. The concentration of each protein was 10 μM. The recovery curves of the fluorescence signals represent the averaged signals from three droplets, and data are presented as means ± SD. (E) Left, representative SDS-PAGE of sedimentation-based assays showing the amount of PSD-95 and IRSp53 recovered from the condensed phase or pellet (P) and the aqueous phase or supernatant (S) at indicated protein concentrations. Right, quantification of protein distribution in E. Results are from three independent batches of experiments and are presented as mean ± SD. (F) Left, sedimentation experiments showing the distribution of PSD-95 and IRSp53 in P and S at indicated protein concentrations. Right, quantification of protein distribution in F. Results are from three independent batches of experiments and are presented as mean ± SD. (G) Fluorescence images showing truncations of PSD-95 with weakened LLPS capability with IRSp53. The N-terminal 30 residues proceeding PDZ1 (NT in A) are not required for phase separation. (H) Fluorescence images showing truncations of IRSp53 with weakened LLPS capability with PSD-95. Replacement of the BAR domain with Myo-CC retained its phase separation with PSD-95. Source data are available for this figure: SourceData F1.

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