Membrane composition and curvature in SNX9-mediated actin polymerization

TY - JOUR

T1 - Membrane composition and curvature in SNX9-mediated actin polymerization

AU - Vaishnav, Pankti

AU - Kondo, Hanae Shimo

AU - Gadsby, Jonathan R

AU - Blake, Thomas C A

AU - Dobramysl, Ulrich

AU - Mason, Julia

AU - Atherton, Joseph

AU - Gallop, Jennifer L

PY - 2025/5/1

Y1 - 2025/5/1

N2 - 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.

AB - 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.

KW - Sorting Nexins/metabolism

KW - Humans

KW - Actins/metabolism

KW - Polymerization

KW - Phosphatidylinositol 4,5-Diphosphate/metabolism

KW - Phosphatidylinositol Phosphates/metabolism

KW - Liposomes/metabolism

KW - Cell Membrane/metabolism

KW - Phosphatidylinositols/metabolism

KW - Actin-Related Protein 2-3 Complex/metabolism

KW - Protein Binding

KW - Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism

KW - Actin Cytoskeleton/metabolism

UR - http://www.scopus.com/inward/record.url?scp=105003169029&partnerID=8YFLogxK

U2 - 10.1091/mbc.E24-09-0419

DO - 10.1091/mbc.E24-09-0419

M3 - Article

C2 - 40105919

SN - 1059-1524

VL - 36

SP - ar54

JO - Molecular biology of the cell

JF - Molecular biology of the cell

IS - 5

ER -