Figure 1. Eps15 and Fcho1 assemble into protein-rich domains on membrane surfaces.

(a-d) Center slices (top) and corresponding z-projections (bottom) of representative GUVs incubated with 500 nM of the indicated protein(s): Atto-594-labeled Fcho1 and CF488a-labeled Eps15 variants. Eps15 variants contain an N-terminal 6xHis tag. GUVs contain 79% DOPC, 15% DOPS, 5% PtdIns(4,5)P₂, and 1% DPEG10-biotin unless otherwise indicated. (a) Full-length Fcho1 alone on GUVs, (left) and full-length Eps15 alone on GUVs containing 97% DOPC, 2% DOGS-NTA-Ni, 1% DPEG10-biotin (center). Cartoons (right) depict domain organization of Fcho1 and Eps15 dimeric forms. (c) GUVs incubated with both Fcho1 and Eps15 display a single protein-rich domain or (d) several protein-rich domains. (e) Two representative center slices of GUVs bound with Eps15 lacking the 6x histidine tag and Fcho1, which co-assemble into protein-rich domains on the membrane (left). Domains were observed on 83 ±2% (SEM) of GUVs (49 GUVs, n=4 biologically independent experiments). (f) GUVs labeled with 0.1% Texas Red-DHPE lipid were incubated with 500 nM each of CF488a-labeled Eps15 and unlabeled Fcho1. (g) Frequency of GUVs displaying protein-rich domains for each set of proteins. GUVs were counted as displaying protein-rich domains if they contained distinct regions in which protein signal intensity differed by at least 2-fold and the bright region covered at least 10% of the GUV surface in any z-slice. For each bar, n=4 biologically independent experiments with at least 40 total GUVs for each condition. All GUV experiments were conducted at room temperature, 22°C. Scale bars are 5 μm. Data are presented as mean ± SEM. See Source Data Figure 1.