Fig 1: Effect of AP3D1 mutations on AP3 complex function.(A) Mutations in AP3D1 that abolish interaction with ARF1 sites relocalize δ-WT-SG to the cytosol. Lattice-SIM imaging of HeLa Cas9 cells expressing δ-WT-SG, δ-ARF1Site1 + Site2-SG (δF77S, δM110S, δL111S + δH157D, δK159D, δR187D, and δR163D), δ-ARF1Site1-SG (δF77S, δM110S, and δL111S) or δ-ARF1Site2-SG (δH157D, δK159D, δR187D, and δR163D). Endogenous δ was abolished using transient CRISPR-KO. Schematics indicate mutated ARF1-binding sites as red crosses. Scale bars, 10 μm. (B) Average number of δ-SG positive structures per cell. Three independent experiments were performed (10 to 15 cells quantified per condition). Tukey’s multiple comparisons test (post hoc, FWER = 0.05) was performed. ***P ≤ 0.001. (C) Schematics indicate charge pairs between neighboring AP3 complexes involved in stripe formation for WT AP3 and mutated δ. Mutations in δ that abolish interaction with AP3M1 and AP3B1 subunits affect endosomal/vesicular dynamics and shape. Lattice-SIM imaging of HeLa Cas9 cells expressing δ-WT-SG, δ-μ3 + δ-β3-SG (δE313K, δD314K, δD316K + δK342D, δK371D, δK378D, δK379D, and δK386D), δ-μ3-SG (δE313K, δD314K, and δD316K), or δ-β3-SG (δK342D, δK371D, δK378D, δK379D, and δK386D). Endogenous δ was abolished using transient CRISPR-KO. Scale bars, 10 and 5 μm in expanded insets. Blue arrows indicate tubular shaped δ-WT-SG structures. (D and E) Circularity (D) and mobility (E) of δ-WT-SG–positive dots determined from (C). Circularity was calculated with Amaris software. Mobility was established on ~100 puncate. Tukey’s test was performed. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; n.s., not significant. (F) Schematic of Flow cytometry-based AP3 trafficking assay. (G) Flow cytometry analysis of GFP-mCh-LAMP1-RUSH trafficking to the lysosome. Endogenous δ was depleted in cell lines expressing δ-WT-Halo mutants. Lysosomal arrival is measured by quenching of the GFP signal after 1-hour biotin incubation (GFP/mCherry ratio). A minimum of ~1000 cells were analyzed per condition. Tukey’s test was performed. **P ≤ 0.01 and ***P ≤ 0.001.