Skip to main content
. Author manuscript; available in PMC: 2017 Aug 21.
Published in final edited form as: Cell. 2016 Aug 25;166(5):1163–1175.e12. doi: 10.1016/j.cell.2016.07.008

Figure 4. Phase Transition of the SynGAP CC-PBM/PSD-95 PSG Complex.

Figure 4

(A) Isolated SynGAP CC-PBM and PSD-95 PSG solutions are stable and homogeneous at 100 µM concentration under light microscope at room temperature (RT). Mixing the two proteins, each at a concentration of 100 µM, resulted in formation of numerous droplets. The images shown in the figure were acquired 3 min and onward after mixing. The dashed box is the region of zoomed-in analysis in (B).

(B) The small droplets underwent time-dependent coalescence into larger ones.

(C) Schematic diagram illustrating the sedimentation assay to separate the condensed liquid phase and the aqueous phase of the SynGAP CC-PBM/PSD-95 PSG mixtures in the rest of the study.

(D) Representative SDS-PAGE analysis and quantification data showing the distribution of proteins between aqueous-solution/supernatant (S) and condensed liquid phase/pellet (P) fractions for various SynGAP CC-PBM/PSD-95 PSG mixtures. The concentration of each protein in all of the experiments is at 100 µM, calculated as their monomer units.

(E) Sedimentation assay showing that the phase transition of the SynGAP/PSD-95 complex is concentration dependent. SynGAP and PSD-95 were mixed at a 1:1 ratio at various concentrations, and the proteins were quantified by their band intensities.

(F) Time-lapse DIC images of SynGAP CC-PBM/ PSD-95 PSG mixture (1:1 at 100 µM) showing numerous droplets at RT in a coverslip chamber. Droplets are rapidly dispersed after adding the 15AA peptide (see also Movie S1). The arrow refers to the time point of adding the 15AA peptide to the mixture.

(G) SDS-PAGE analysis and quantification results showing pre-formed SynGAP/PSD-95 droplets can be reversed to aqueous phase by the competing 15AA peptide. The indicated concentrations of the peptide or a peptide-free buffer was added to a 1:1 SynGAP/PSD-95 mixture at 100 µM.

(H) DSG-mediated cross-linking reveals PSD-95 PSG undergoes a SynGAP peptide binding-induced dimerization. The CRIPT peptide does not induce dimer formation of PSD-95 PSG. The peptide to PSD-95 molar ratio and reaction time are indicated in the figure.

All statistic data in this figure represent the results from three independent batches of experiments and are expressed as mean ± SD.