Molecular mechanisms of Sorting nexin-9 in membrane triggered actin polymerisation

Abstract

Sorting nexin-9 (SNX9) is a SH3-PX-BAR domain-containing adaptor protein, func- tionally implicated in regulating actin polymerisation in clathrin mediated endocy- tosis and filopodia formation. Alterations in SNX9 expression correlate with several stages of breast cancer metastasis, and poor patient survival in colorectal and prostate cancer. In addition to this, SNX9 is also required for Chlamydia trachomatis entry into cells and contributes to aberrant actin assemblies in a rare disease of phospho- inositide lipid metabolism, Lowe syndrome. It’s multifaceted nature is attributed to its structure. The SH3-PX-BAR domains of SNX9 are able to integrate inter- actions with phosphatidylinositol phosphates (PIPs) at membranes, and membrane curvature sensing to trigger actin polymerisation via the Arp2/3 complex. While membrane binding is key in in vitro purified reconstitutions of actin polymerisation, the lipid specificity and contribution of each membrane domain is under dispute and not well understood in vivo. Three models have been proposed for SNX9-membrane binding: (1) broad non- specific interactions with diverse phosphoinositides (PIPs), (2) the coincidence of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) and phosphatidylinositol-(3)-pho- sphate (PI(3)P) each binding the BAR and PX-domains respectively or, (3) a particu- lar specificity for phosphatidylinositol-(3,4)-bisphosphate (PI(3,4)P2). To distinguish between these models, I used Surface Plasmon Resonance (SPR) and Bio-Layer Inter- ferometry (BLI) to determine the membrane specificity of SNX9. Both SPR and BLI studies confirm a SNX9 binding preference for PI(4,5)P2/PI(3)P at low SNX9 con- centrations whereas a more nuanced picture emerges at higher concentrations, where more SNX9 binds to PI(4,5)P2/PI(3)P liposomes. The affinity, however, is similar to the other compositions, suggesting SNX9 can assemble differently depending on PIPs present in the liposomes. Notably, SNX9 binding to PI(4,5)P2/PI(3)P liposomes is inhibited by excess PI(3)P, indicating a potential regulatory mechanism where local PI(3)P concentrations control SNX9 recruitment. Preliminary Cryogenic Electron Microscopy (cryo-EM) analysis revealed that SNX9 forms a protein coat on PI(4,5)P2/PI(3)P liposomes and deforms the membrane forming tubules in both PI(4,5)P2/PI(3)P and PI(3,4)P2 compositions at high SNX9 concentrations. Investigation of SNX9 membrane binding domains in Xenopus egg extract-based actin polymerisation assays demonstrates that PX-BAR domains are es- sential for two distinct actin architectures. To visualise this, I used Cryogenic Electron Tomography (cryo-ET) to study the actin nucleating from 10% PI(4,5)P2 supported lipid bilayers and 100 nm PI(4,5)P2/PI(3)P liposomes, both of which require SNX9. Despite this, the actin architectures are distinct from the different compositions. The work in this thesis provides insights into how SNX9 localisation can be regulated by the membrane and its composition, allowing it to be present at two distinct actin architectures that are nucleated by similar sets of actin regulators. This membrane- dependent regulation enables precise spatiotemporal control of SNX9-mediated actin assembly