Vaishnav Pankti, Kondo Hanae Shimo, Gadsby Jonathan R, Blake Thomas C A, Dobramysl Ulrich, Mason Julia, Atherton Joseph, Gallop Jennifer L
Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, United Kingdom.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
Mol Biol Cell. 2025 May 1;36(5):ar54. doi: 10.1091/mbc.E24-09-0419. Epub 2025 Mar 19.
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)P) and phosphatidylinositol (3)-phosphate (PI(3)P) compared with phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P), 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.
分选连接蛋白9（SNX9）是一种膜结合支架蛋白，它有助于病毒摄取和炎症反应，并且与多种癌症的不良预后相关。它参与表皮生长因子受体、β1整合素和膜型1基质金属蛋白酶的内吞作用以及线粒体衍生小泡的形成。SNX9的Bin-发动蛋白-衔接蛋白-反转（BAR）-PX（Phox同源）结构域结合磷酸肌醇脂质，而Src同源3（SH3）结构域与发动蛋白和神经维斯科特-奥尔德里奇综合征蛋白（N-WASP）相互作用，以刺激Arp2/3复合物介导的肌动蛋白聚合。在这里，我们使用生物层干涉术、无细胞重组和超分辨率显微镜来分析SNX9在膜上的特异性和活性。我们发现，尽管亲和力相似，但与磷脂酰肌醇（三磷酸）（PI(3,4)P）相比，更多的SNX9能够结合含有磷脂酰肌醇（4,5）-二磷酸（PI(4,5)P）和磷脂酰肌醇（3）-磷酸（PI(3)P）的脂质体。肌动蛋白组装需要PX-BAR和SH3相互作用网络。对丝状伪足样重组体进行的三维直接随机光学重建显微镜观察表明，SNX9和Cdc42依赖性肌动蛋白组装相关蛋白转导子1（TOCA-1）在肌动蛋白掺入位点既能形成扁平组装体，也能形成约0.5μm的弯曲组装体。最后，使用冷冻电子断层扫描技术，我们表明SNX9构建了分支状和束状肌动蛋白网络，证明了其在肌动蛋白重塑中发挥多功能作用的潜力。
