Panel A: Diagram of various CAST protein constructs. The constructs are labeled as CAST 7RKA, CAST 1-400, CAST 401-957, CAST ΔCR, CAST 7RKA-delta CR, and CAST 7RKA+6R9KA (CASTmt). Panel B: Sequence alignment showing conserved regions of CAST with mouse ELKS, Bruchpilot, and elks-1. The conserved amino acids are highlighted. Panel C: Fluorescence microscopy images showing EGFP-Endophilin-A2 expressed with mCherry-CAST constructs. The images display condensates formed by different CAST constructs. The right panel includes dot plots showing the ratio of average fluorescence intensity at the condensate to the cytosolic region. Panel D: Western blot analysis showing the affinity of mutant CAST for Endophilin-A-1. The bar graph quantifies the binding affinity of different CAST constructs to Endophilin-A-1.
Determining the amino acid region of CAST that binds to endophilin-A. (A) Cartoon image of the deletion and mutation constructs of CAST. (B) The regions of the amino acids sequence that have been evolutionarily conserved in CAST. The amino acids 6–42 of mouse CAST shown in the upper panel are partially homologous to Bruchpilot, the ortholog of Drosophila. The 155–212 amino acids of mouse CAST shown in the panel below are partially homologous not only to Bruchpilot but also to elks-1, a homologous molecule in C. elegans. (C) EGFP–endophilin-A2 expressed with mCherry-CAST, CAST-7RKA, or CAST-ΔCR results in condensates, which are rarely observed with CAST-7RKA+ΔCR and CAST-7RKA+6R9KA. The dotted line indicates a line scan of each fluorescence protein. Right dot plots show the ratio of average fluorescence intensity at the condensate (B) to cytosolic region (A). Scale bar: 10 μm. n = 10 cells, each. P values were obtained by comparison with CAST-WT (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, ****P < 0.0001). (D) Decrease the affinity of mutant CAST for endophilin-A-1. MBP-CAST-1-400, MBP-CAST-1-400-7RK+6R9KA, or MBP-CAST-1-400-7RK+ΔCR were precipitated with GST–endophilin-A1-full. MBP-CAST-1-400 was detected by the western blot analysis and GST–endophilin-A1-full was detected by the CBB staining. Right bar graph shows quantification of the left bands (n = 3 independently prepared samples). Results are mean ± SD (n = 3). P values were obtained by comparison with CAST-WT (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, **P < 0.01). Source data are available for this figure: SourceData F6.