Membrane composition and curvature in SNX9-mediated actin polymerization

Membrane-binding adaptor protein Sorting nexin 9 (SNX9) contributes to viral uptake and inflammation and is associated with worse outcomes in several cancers. It is involved in endocytosis of epidermal growth factor receptors, β1-integrin and membrane type 1 matrix metalloprotease and in mitochondrial-derived vesicles. Its BAR-PX domain binds phosphatidylinositol phosphates (PIPs) and its SH3 domain interacts with dynamin and N-WASP to stimulate Arp2/3 complex actin polymerization during vesicle scission. Recent complexities have arisen in SNX9’s lipid specificity and its involvement both endocytic and filopodial membrane topologies. Here we use biolayer interferometry, cell-free reconstitution and superresolution microscopy to analyse the activities of SNX9. We find that more SNX9 binds membranes that contain PI(4,5)P2 and PI(3)P compared with PI(3,4)P2, despite having similar affinity, suggesting SNX9 can assemble into different macromolecular arrangements depending on the composition. Actin assembly requires the wider protein and lipid network provided by PX-BAR and SH3 interactions. 3D direct stochastic optical reconstruction microscopy on filopodia-like reconstitutions shows that SNX9 and related protein Transducer of Cdc42 activation-1 (TOCA-1) are competent to form both tubular and plaque-like organizations with the actin machinery. Finally, using cryo-electron tomography we show that SNX9 assembles both branched and bundled actin filaments demonstrating its multifunctional nature.