Significance Statement
SNX9 is involved in actin assembly during cell migration, endocytosis, and disease. It is unclear how SNX9 links membrane binding to actin architecture.
Using binding measurements and a cell-free system, the authors find differences between phosphatidylinositols and show that membrane binding is needed for actin assembly. Superresolution microscopy reveals that SNX9 and a related protein, TOCA-1, can attain curved and flat orientations. Cryo-electron tomography is used to show both branched and bundled actin filaments arise downstream of SNX9.
The work shows how the assembly of SNX9 makes diverse actin networks.
Abstract
Sorting nexin 9 (SNX9) is a membrane-binding scaffold protein that 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 formation of mitochondrial-derived vesicles. The SNX9 Bin-Amphiphysin-Rvs (BAR)-Phox homology (PX) domains bind phosphoinositide lipids and the Src homology 3 (SH3) domain interacts with dynamin and Neural-Wiskott Aldrich syndrome protein (N-WASP) to stimulate Arp2/3 complex–mediated actin polymerization. Here we use biolayer interferometry, cell-free reconstitution, and superresolution microscopy to analyze the specificity and activities of SNX9 at membranes. We find that more SNX9 can bind liposomes containing phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) and phosphatidylinositol (3)-phosphate (PI(3)P) compared with phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), despite similar affinities. Actin assembly requires the network of both PX-BAR and SH3 interactions. Three-dimensional direct stochastic optical reconstruction microscopy on filopodia-like reconstitutions shows that SNX9 and related protein transducer of Cdc42-dependent actin assembly-1 (TOCA-1) can form both flat and ∼0.5 µm curved assemblies at actin incorporation sites. Finally, using cryo-electron tomography, we show that SNX9 builds both branched and bundled actin networks demonstrating its potential for multifunctional roles in actin remodeling.
