CNRS UMR7654, Institut Polytechnique de Paris, 91120 Palaiseau, France.
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91190 Gif-sur-Yvette, France.
Int J Mol Sci. 2021 Apr 16;22(8):4115. doi: 10.3390/ijms22084115.
During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid-liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.
在细胞迁移过程中，前缘的突出是由 Arp2/3 依赖性分支肌动蛋白网络的聚合驱动的。Arp2/3 抑制蛋白 Arpin 负调控迁移的持久性。为了更好地理解细胞中 Arpin 的调节作用，我们寻找了它的相互作用伙伴，并使用酵母双杂交筛选和全长 Arpin 作为诱饵的共免疫沉淀，鉴定了 Tankyrase 1 和 2（TNKS）。Arpin 通过其酸性尾部的一个 C 末端结合位点与 TNKS 的锚蛋白重复结构域相互作用，该结合位点与 Arp2/3 结合位点重叠。发现 Arpin 过表达时会溶解 TNKS 的液-液相分离。为了分离 Arpin 与 TNKS 和 Arp2/3 的相互作用，我们在 Arpin 尾部引入了点突变，并尝试使用随机质粒整合或在 基因座上进行补偿性敲入，来挽救 Arpin 敲除细胞中迁移持久性增加的现象。影响与 Arp2/3 或 TNKS 相互作用的 Arpin 突变不足以完全消除 Arpin 的活性。只有影响这两种相互作用的突变才使 Arpin 完全失活，这表明存在两种独立的途径，Arpin 通过这两种途径控制迁移的持久性。
