Fig. 5: Liquid-Liquid Phase Separation (LLPS) of quaternary USH2 protein condensate.
From: Temporal and spatial assembly of inner ear hair cell ankle link condensate through phase separation
a Co-sedimentation-based assay showing that proteins were much more enriched in the pellet in WHRN/USH2A-CT than the isolated group. b Fluorescence images showing that droplets formed in WHRN/USH2A-CT but not the isolated group (20 μM each). WHRN and USH2A-CT were labeled with Alexa 488 and Cy3, respectively. Labeled proteins were added at a ratio of 1%. Scale bar: 5 μm. c Summaries of the stoichiometry of WHRN, WHRN NTD, WHRN △NTD, USH2A-CT, or USH2A-CT(S). d Co-sedimentation-based assay showing that the pellet enrichments of the two proteins in WHRN/USH2A-CT were deceased when USH2A-CT △PBM was introduced. Proteins were mixed at the final concentration of 20 μM each. e Co-sedimentation-based assays showed that the pellet enrichments of the two proteins in WHRN/USH2A-CT were decreased when WHRN △NTD or USH2A- CT(S) was introduced. Proteins were mixed at the final concentration of 20 μM each. f Fluorescence images show that the droplets formed by WHRN/USH2A-CT decreased in size or number when USH2A-CT △PBM, USH2A-CT(S), or WHRN △NTD was introduced. Proteins were mixed at the final concentration of 20 μM each. Scale bar: 10 μm. g Fluorescence images showed that the droplets were colocalized in highly enriched WHRN, USH2A-CT, PDZD7, and ADGRV1-CT. WHRN, USH2A-CT, PDZD7, and ADGRV1-CT were labeled with Alexa 488, Cy3, Cy5, and Alexa-405, respectively. Labeled proteins were added at a ratio of 1%. Scale bar: 5 μm. h Kinetic quantification of the FRAP assay showing the recovery rates of WHRN, USH2A-CT, ADGRV1-CT, and PDZD7. Mean ± SD, n = 3–5. WHRN NPDZ12 and PDZD7 were abbreviated as WHRN and PDZD7, respectively. △PBM or △NTD, the truncation of PBM or NTD. PBM, PDZ binding motif. NTD, N-terminal domain. Source data are provided as a Source Data file.
