黏着斑相关蛋白Rsu1及CLASP2动态调控细胞骨架的分子机制研究

黏着斑是连接细胞骨架与细胞外基质的重要结构，对于细胞的粘附和迁移等过程至关重要。黏着斑结构具有高度动态性。在细胞迁移过程中，黏着斑经历形成、成熟、解聚和再形成的周转过程，以调控细胞的迁移方向和速度。黏着斑周围的微丝和微管在促进黏着斑周转过程中发挥着重要的作用。因此，细胞骨架的动态调控对于黏着斑的动态至关重要。黏着斑相关蛋白包含黏着斑核心蛋白和黏着斑周边蛋白，这些蛋白一起构成庞大的黏着斑网络，对黏着斑的组装、成熟和解聚的过程，以及黏着斑周围细胞骨架的调控过程至关重要。Rsu1是黏着斑核心蛋白之一，通过与PINCH1的相互作用定位于黏着斑上。Rsu1与PINCH1可发挥协同作用，调控黏着斑的动态结构和应力纤维的形成。另一方面，黏着斑周边蛋白ELKS、LL5β定位于黏着斑周围，可与微管末端追踪蛋白CLASP相互作用，进而调控微管在黏着斑处的靶向。然而，这些蛋白在黏着斑周围细胞骨架调控中具体的分子机制仍不清楚。本论文第一部分中，为了研究黏着斑核心蛋白Rsu1与PINCH1调控黏着斑动态，以及应力纤维形成的分子机制，利用蛋白质相互作用实验，确定了Rsu1与PINCH1具体的结合位置，并进一步获得了该复合物的晶体结构。通过结构分析，发现PINCH1上Rsu1的结合表面与微丝的结合表面相互重叠。因此，推测Rsu1可能通过结合PINCH1，破坏PINCH1与微丝的结合，进而抑制IPP介导的对微丝捆绑成束的功能。通过体外的微丝捆绑实验以及细胞实验，证明了Rsu1与PINCH1的相互作用能够抑制IPP介导的微丝捆绑，从而影响黏着斑的动态与应力纤维的组装。此外，发现了细胞内适量的Rsu1有助于IPP复合物的稳定，而帮助黏着斑附近微丝的捆绑。最后探讨了不同细胞类型中Rsu1对细胞迁移的功能，这些功能可能与Rsu1的相对表达水平紧密相关。综上，过量的Rsu1破坏了PINCH1与微丝的结合，从而抑制黏着斑周围微丝的捆绑作用。与此相反，适量的Rsu1有利于IPP复合物的稳定，从而促进微丝的捆绑。Rsu1的这种双重的功能使之能够调控黏着斑的动态以及应力纤维的形成，从而调控细胞的迁移。本文第二部分中，为了研究黏着斑周边蛋白ELKS、LL5β与CLASP调控微管正末端靶向黏着斑的分子机制，通过生化实验确认了CLASP2/CLIP170、CLASP2/LL5β以及ELKS2/LL5β之间的具体结合位置，并成功获得了这些复合物的晶体结构。通过结构分析，结合蛋白相互作用实验，设计了突变以打破这些蛋白质之间的结合。在细胞内，发现CLIP170以及ELKS1能够发生液-液相分离而形成凝聚体，并且将CLASP2招募到其中。有趣的是，CLASP2能够引起CLIP170以及ELKS1凝聚体发生稳定的接触，从而帮助微管靶向至黏着斑。此外，发现CLASP2的磷酸化调节CLIP170凝聚体与ELKS1凝聚体的分离，从而使微管末端离开黏着斑。综上，CLASP2通过调控凝聚体之间的相互作用，调控了微管正末端与黏着斑的接触与分离。通过以上两部分的研究，利用了生化实验、结构分析以及细胞实验，阐释了Rsu1和CLASP2等蛋白调控黏着斑周围细胞骨架的分子机理，为进一步深入理解黏着斑及其周围的细胞骨架的动态调控提供了新的思路和方向。

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