Extended Data Fig. 1: Supporting data for Fig. 1.
(a) Alignment of muniscin APA domains from four model organisms. Vertebrates (H. sapiens, D. rerio) contain two homologs of FCHo and the neuronal SGIP1. D. Melanogaster and C. elegans each contain a single muniscin. Black boxes: 100% identity, full color: 100% similarity, partial color: >75% similarity. (b) Confocal slices of AP2core bound to lipid-coated beads alone or pre-seeded with muniscin. Both proteins are added at 50 nM concentration. Scale bar 5 µm. FCHoBAR and FCHoBAR+APA images are also used in Fig. 1f. (c) Maximum intensity projections of AP2core (blue) and FCHoBAR+APA (green) bound to beads. Both proteins are added at 50 nM concentration. (d) Representative images of three assay conditions: AP2 alone, AP2 + FCHoBAR, and AP2 + FCHoBAR+APA. (e) Puncta formation of AP2core in the presence of soluble SGIPAPA at three concentrations. AP2 is held at 2 µM concentration. (f) Representative images of AP2 binding to SLBs +/- SGIPMP+APA, which includes the membrane phospholipid-binding domain (MP) and APA domain of SGIP. (g) Quantification of SLBs showing that AP2 binding is increased by the addition of 50 nM SGIPMP+APA. (h) Quantification of SLBs showing that the number of AP2 puncta per bead is increased by the addition of 50 nM SGIPMP+APA. (G-H) Data are mean +/- sd of triplicate measurements. ****P < 0.0001 for unpaired t-test (i) Representative pulldown of AP2 by either HT:SGIPAPA, HT:SGIPMP+APA, or HT:FCHo2BAR+APA in the presence of 5 µM heparin. HT: HaloTag. (j) Representative full SDS-PAGE gels of the protease sensitivity experiments quantified in Fig. 1g. Data for graphs in g,h are available as source data.
