Journal: Nature structural & molecular biology

Article Title: Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization

doi: 10.1038/s41594-022-00749-z

Figure Lengend Snippet: a. AP2 comprises four subunits; alpha (α), beta (β2), mu (μ2), and sigma (σ2). Activation of AP2 (→) exposes binding sites for cargo (C) and membrane (M). b. Domain structure of muniscins. Dashed boxes highlight purified constructs. MP: Membrane-phospholipid-binding domain. c. Confocal microscopy of AP2core bound to SLB-coated beads. Scale bar 5 μm Right: AP2core adsorption on SLBs, normalized to FCHo2. N > 50 beads. d. Maximum intensity projections of AP2core bound to beads. Right: Quantification; N > 100 puncta over N > 30 beads. c,d. Box and whisker plots show the mean (line), 25th and 75th percentiles (box), and the min and max (bars). ****P < 0.0001 for unpaired t-test. e. Representative pulldowns of AP2 by HT:SGIPAPA. HT: HaloTag. f. Binding affinity of SGIPAPA for AP2, determined using microscale thermophoresis. N = 2 independent experiments. g. Protease sensitivity assay. Left: Representative SDS-PAGE gel. Right: Quantification of μ2 protease sensitivity. N = 3 independent experiments. Error bars represent SEM. Uncropped images for e,g and data for graphs in d,f,g are available as source data.

Article Snippet: 80 pmol of HaloTag bait protein or HaloTag control was combined with 40 pmol of AP2 core prey and 10 μL of Magne HaloTag Bead slurry (20%, Promega) in TBST with 1 mM DTT (250 μL total volume for each reaction).

Techniques: Activation Assay, Binding Assay, Membrane, Purification, Construct, Confocal Microscopy, Adsorption, Whisker Assay, Microscale Thermophoresis, Sensitive Assay, SDS Page

Journal: Nature structural & molecular biology

Article Title: Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization

doi: 10.1038/s41594-022-00749-z

Figure Lengend Snippet: (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. (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.

Article Snippet: 80 pmol of HaloTag bait protein or HaloTag control was combined with 40 pmol of AP2 core prey and 10 μL of Magne HaloTag Bead slurry (20%, Promega) in TBST with 1 mM DTT (250 μL total volume for each reaction).

Techniques: Concentration Assay, Binding Assay, Membrane, SDS Page

Journal: Nature structural & molecular biology

Article Title: Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization

doi: 10.1038/s41594-022-00749-z

Figure Lengend Snippet: a. Representative pulldowns of AP2 by various HT:D/ExxxLL cargos or HT:YxxΦ-cargo in the presence or absence of 5 μM SGIPAPA (left). All samples contained 5 μM heparin. b. Representative pulldowns of AP2 by HT:SGIPAPA ± YxxΦ-cargo peptide. HT: Halotag. Shading represents a 95% confidence interval for each curve. c. Binding affinity of SGIPAPA to AP2 was determined by microscale thermophoresis with YxxΦ-cargo peptide. d. Left: Representative SDS-PAGE showing protease sensitivity of μ2 induced by SGIPAPA ± YxxΦ-cargo peptide. Right: Quantification of μ2 protease sensitivity induced by SGIPAPA with various amounts of YxxΦ-cargo peptide. Uncropped images for a,b,d and data for graphs in c,d are available as source data.

Article Snippet: 80 pmol of HaloTag bait protein or HaloTag control was combined with 40 pmol of AP2 core prey and 10 μL of Magne HaloTag Bead slurry (20%, Promega) in TBST with 1 mM DTT (250 μL total volume for each reaction).

Techniques: Binding Assay, Microscale Thermophoresis, SDS Page

Journal: Nature structural & molecular biology

Article Title: Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization

doi: 10.1038/s41594-022-00749-z

Figure Lengend Snippet: (A) Representative pulldown of AP2 by HT:SGIPAPA in the presence of 5 μM heparin and either YxxΦ or ExxxLL cargo peptide. HT: HaloTag. (B) Representative pulldown of AP2 by either HT:SGIPAPA or HT:FCHo2BAR+APA in the presence of 5 μM heparin and YxxΦ-cargo peptide. HT: HaloTag.

Article Snippet: 80 pmol of HaloTag bait protein or HaloTag control was combined with 40 pmol of AP2 core prey and 10 μL of Magne HaloTag Bead slurry (20%, Promega) in TBST with 1 mM DTT (250 μL total volume for each reaction).

Techniques: