Talin激活机制及其力学调控

Talin is a high molecular weight and highly abundant cytosolic protein, which regulates a wide variety of integrin-mediated cell adhesion processes, such as cellular spreading, migration, proliferation and the cellular deformation-related processes. Talin has been known to not only be a mechanical linker between cell-extracellular matrix and the actin cytoskeleton, but active integrin inside-out mechanical signaling, thereby form the bidirectional mechanical signaling pathway. Talin adopts an auto-inhibited conformation in its inactive state, where the autoinhibited interaction between F3 domain and ROD domain shields the F3 domain of talin and prevents it interacting with integrin. Uncontrolled talin activity could result in dysfunction of integrin, and lead to many cell function disorders. Therefore, activation mechanism of talin is investigated widely in recent decades, but is not compelete and convinced untill today. In this study, we will make use of the self-made Atomic Force Microscope to perform the single molecule force experiments to investigate the interactions between F3 and ROD domain by measuring their interaction, employ the molecular dynamics method to carry out the all-atomic simulation to study the corresponding disassociation process of F3 and ROD domain of talin. The interaction between F2F3 domain and phospholipid bilayer also will be studied in absence and presence of ROD domain using both AFM and molecular dynamics methods, where three kinds of phospholipid bilayer with different component will be considered. The dynamics properties and atom-level information about talin autoinhibition relief would be obtained by these experiments. Combining the experimental and simulational results, we could find out the key reisudes responsible for the disassociation processes of F3-ROD and F2F3-membrane, and conclude the effect of membrane and mechanical force on talin activation. Our research will supply the powerful evidences to improve the activation mechanism of talin and its mechanical force regulation, enrich the mechanism of intercellular mechanical signaling, and provide the theoretical foundations for treating the disease involving integrin-mediated cell adhesion.

踝蛋白(talin)参与诸多整合素相关的细胞粘附行为，可将细胞外基质和细胞内骨架肌动蛋白通过整合素串联起来，是细胞间力信号传导通路中的重要一环。在非活化状态下，talin的F3域和ROD域相互作用，构成自抑制结构，其活化后可发挥功能，但无控制的活化又会导致整合素功能紊乱，引起诸多疾病，因此talin的激活机理受到广泛关注，但至今没有形成完整结论。本项目将通过单分子力学实验研究talin的F2F3域与ROD域的相互作用，F2F3域与不同组分磷脂膜的作用，以及F2F3域在受到ROD域竞争作用时与不同组分磷脂膜的相互作用，并使用分子动力学方法模拟相应过程，得到对应的力谱和原子级别的信息。综合力谱实验与理论模拟的结果，确定talin活化过程中的关键残基，明确磷脂膜与机械力在此过程中的作用，总结出talin的激活及力学调控机制。本研究将有助于完善细胞间力信号传导机理，为治疗相关疾病提供理论依据。

踝蛋白(talin)参与诸多整合素相关的细胞粘附行为，可将细胞外基质和细胞内骨架肌动蛋白通过整合素串联起来，是细胞间力信号传导通路中的重要一环。在非活化状态下，talin的F3域和ROD域相互作用，构成自抑制结构，其活化后可发挥功能，但无控制的活化又会导致整合素功能紊乱，引起诸多疾病，因此talin的激活机理受到广泛关注，但至今没有形成完整结论。. 本项目首先利用单分子力谱与分子动力学模研究了talin的F2F3域与RS域的自抑制相互作用。我们的研究发现：离子浓度的升高会减弱F2F3域和RS域相互作用的强度；D1676、D1763、Q1774和E1805在F2F3域和RS域相互作用过程中起关键作用，其中D1676A和E1805A可以明显的影响F2F3域和RS域的断键力；野生型的F2F3域与RS域的相互作用的寿命呈弱逆锁键的性质(在低力时变化不显著，在高力时明显减低)，原因是外力可以调节自抑制相互作用在两种不同构象状态下的平衡。这些发现揭示了talin激活的负调节机制，并为黏着斑中talin的功能提供了新的视角。同时，我们将磷脂膜考虑进来，发现了负电磷脂PS可以轻微的增强F2F3域与磷脂双分子层的相互作用，F1域与膜的相互作用可以增强talin头部与膜的结合强度，我们推测F1F2F3三个结构域与负电磷脂的相互作用的吸引力大于与解离RS的力，进一步验证了我们的talin激活的负调节机制。另外，对于磷脂膜的动态性质我们也做了初步的探讨，发现了胆固醇浓度对磷脂膜的厚度，面积，以及透水性都有显著的影响；力致的磷脂膜变形可以引起不同磷脂的聚集情况。. 我们的研究确定talin活化过程中的关键残基，明确磷脂膜与机械力在此过程中的作用，总结出talin的激活及力学调控机制。本研究将有助于完善细胞间力信号传导机理，为治疗相关疾病提供理论依据。