訊息互擾與模組協作：使用合成阻動篩選揭示了兩種不同的細胞骨架重塑模式

Abstract

在我們的綜合研究中，我們調查了影響細胞骨架重塑和細胞遷移的兩種不同機制。我們最初的工作集中在serine-threonine kinase 40 (STK40)和mitogen-activated protein kinase (MAPK)之間的協同作用，突顯出合成阻動的概念。通過shRNA-藥物的雙重篩選，我們觀察到STK40和MAPK的同時抑制顯著減少了細胞遷移。這種效應主要來自於STK40基因敲除強化了點狀粘附 (focal adhesion, FA)複合體，這種基因敲除降低了yes-associated protein (YAP) 蛋白的穩定性並減少了其核質比。MAPK的抑制進一步損害了YAP的活性，且破壞FA的動態平衡。這些對於STK40-YAP-MAPK路徑的協調調控, 為我們所稱的“合成阻動”提供了基礎，這是一種調節細胞運動性的新策略。接著我們進一步探討從合成阻動篩選中指出的另一對蛋白的交互作用——Ste20-like kinase (SLK)-Rho-associated protein kinase (ROCK)，我們將焦點放在SLK如何以非磷酸化的方式與Ezrin互動並重塑肌動蛋白結構。具體來說，我們證明了在SLK或Ezrin基因敲除下，HaCaT細胞中的收縮性肌動蛋白束會顯著增加，而抑制Ezrin的磷酸化並未增加這些收縮性肌動蛋白束。此外，Ezrin基因敲除引起的收縮性肌動蛋白束的增加可以通過過表達未磷酸化的Ezrin突變體所逆轉，支持SLK-Ezrin的串聯在細胞遷移期間獨立於磷酸化調控去調節收縮性肌動蛋白束。綜上所述，這些研究揭示了兩條獨立的路徑，通過這些路徑可以調節細胞骨架的重塑，每條路徑都具有其自身的重要性，並提供了管理細胞遷移和細胞骨架組織的創新方法。這些發現不僅加深了我們對細胞運動的理解，也為以異常細胞遷移為特徵的疾病引入了潛在的治療目標。

In our comprehensive study, we investigated two distinct mechanisms that influence cytoskeletal remodeling and cell migration. Our initial work focused on the synergistic interaction between serine-threonine kinase 40 (STK40) and mitogen-activated protein kinase (MAPK), highlighting the concept of synthetic dysmobility. Through an shRNA-drug two-hit screen, we observed a significant reduction in cell migration by concurrent inhibition of STK40 and MAPK. This effect primarily stemmed from strengthening of focal adhesion (FA) complexes by STK40 knockdown, which destabilized yes-associated protein (YAP) and reduced its nuclear translocation. MAPK inhibition further impaired YAP activities, leading to disruptions in FA dynamics. These insights into the coordinated STK40-YAP-MAPK pathway provide a basis for what we term “synthetic dysmobility”, a novel strategy to modulate cell motility. We further explored another interaction pair identified from our screen, the Ste20-like kinase (SLK)-Rho-associated protein kinase (ROCK) interaction, focusing on how SLK interacts with Ezrin to remodel actin structures in a particularly phosphorylation-independent manner. Specifically, we demonstrated a significant increase in contractile actin bundles in migrating HaCaT cells under knockdown of SLK or Ezrin, while suppression of Ezrin phosphorylation did not increase those bundles. Moreover, the increase in contractile actin bundles caused by Ezrin knockdown could be reversed by overexpression an unphosphorylated Ezrin mutant, supporting the phosphorylation-independent facet of SLK-Ezrin cascade in regulating of contractile actin bundles during cell migration. Collectively, these studies unveil two separate pathways by which cytoskeletal remodeling can be modulated, each significant in its own right, and provide innovative approaches for managing cell migration and cytoskeletal organization. These findings not only deepen our understanding of cell motility but also introduce potential therapeutic targets for diseases characterized by abnormal cell migration.