Panel A shows a fluorescence recovery with two microscopy sets of images and a line graph. The images display fluorescent intensity of EGFP-Endophilin-A2 and mCherry-CAST before and after photobleaching at different time points. The line graph plots FRAP percentage over time for EGFP-Endophilin-A2 and mCherry-CAST, with the x-axis representing time in seconds and the y-axis representing FRAP percentage. Panel B presents immunofluorescence images of HEK293T cells treated with 1,6-hexanediol, showing the effect on CAST-Endophilin-A assemblies at different time points. Panel C includes immunofluorescence images of CAST and Endophilin-A1 in rat hippocampal neurons, with confocal laser scanning microscopy (CLSM) and stimulated emission depletion (STED) images. It also shows fluorescence intensity profiles, a histogram, and a cumulative distribution of distances from Endophilin-A1 puncta to the nearest active zone center.
CAST–endophilin condensates exhibit liquid-like behavior and localize near active zones. (A) FRAP analysis of liquid condensates in HEK293T cells co-transfected with mCherry-CAST and EGFP–endophilin-A2. The right panels show representative images before and after photobleaching. Scale bar, 2 μm. The left graph shows fluorescence recovery curves for mCherry-CAST (n = 28, red line) and EGFP–endophilin-A2 (n = 28, green line). Error bars represent the SEM. (B and C) Effect of 1,6-hexanediol treatment on CAST–endophilin-A assemblies. HEK293T cells co-transfected with mCherry-CAST and EGFP–endophilin-A2 were treated with 12.5 % 1,6-hexanediol at the indicated time points. Scale bar; 10 μm (C) Immunofluorescence images of CAST (magenta) and endophilin-A1 (green) in cultured rat hippocampal neurons. The upper left panels show confocal laser scanning microscopy (CLSM) images and corresponding STED images. The upper right panel presents an enlarged view of the region indicated by a white box in the STED image. Arrows indicate sites where CAST and endophilin-A1 co-localize. Scale bars: 2 µm for the original images and 500 nm for the enlarged image. The bottom left panel shows fluorescence intensity profiles from the enlarged STED images. The bottom middle and right panels show a histogram and cumulative distribution, respectively, of the distances from individual endophilin-A1 puncta to the nearest active zone (AZ) center (n = 497 puncta from three independent cultures).