Molecular mechanisms of Wnt and mechanical signaling through β-catenin

固体组织的发育和动态平衡取决于生化和机械信号 控制细胞的命运和由此产生的组织中的细胞。保守的β-连环蛋白是关键 Wnt家族分泌的生长因子信号的效应者，它们决定了细胞在 成人的胚胎发生和组织更新，以及通过细胞-细胞连接传递的机械力 在多细胞组织中。我们假设通过β-链接链传递的机械力 信号和细胞-细胞黏附，我们的总体目标是了解 β-连环蛋白的这些双重作用。我们的战略是使用生化、结构和生物物理方法来 解决这些领域的关键知识差距。 在没有WNTS的情况下，β-连环蛋白结合在一个包括轴蛋白的“破坏复合体”中 和腺瘤性息肉病结肠(Apc)，以及使β-连环蛋白磷酸化的激酶； 蛋白酶体对β-连环蛋白的泛素化和破坏。WNT与受体的结合(FZD) 而LRP5/6使FZD能够招募胞浆蛋白DVL，DVL进而与Axin结合并 从而将破坏复合体招募到激活的受体复合体中。这会导致磷酸化。 LRP5/6胞内结构域，它抑制β-连环蛋白的破坏；稳定的β-连环蛋白进入 并激活目标基因。我们将解决这一途径的关键机械方面，而不是 理解：1)分泌配体如何“激活”β-连环蛋白稳定所需的fzd-dvl相互作用 通过与FZD和LRP5/6胞外富含半胱氨酸结构域的相互作用；2)如何激活DVL 招募Axin关闭β-连环蛋白破坏；3)β-连环蛋白破坏复合体如何形成和相互作用 与泛素化/蛋白酶体机制有关；4)APC在β-连环蛋白破坏中的重要作用。 通过细胞-细胞黏附连接(AJ)的力传递需要E-钙粘附素，β-连环蛋白， 以及α-连环蛋白，它与肌动蛋白细丝结合，形成最小的力敏单位。钙粘附素的紧张状态 可以释放β-连环蛋白，并使其移位到细胞核，不依赖于WNT，但与WNT协同 发信号。要想了解这种由张力触发的β-连环蛋白的释放，需要了解力是如何 通过AJ复合体传播。α-连环蛋白在组织上皮组织中还起着中心作用 基于其与纽蛋白的相互作用，上皮蛋白在肿瘤中丢失(EPLIN)，紧密连接(TJ) 闭锁小带蛋白(ZO)-1和afadin，所有这些都结合肌动蛋白并招募其他支架和 信号蛋白。我们将研究：1)力相关的构象地貌和力 α-连环蛋白单独和与其伙伴结合的响应，包括β-连环蛋白如何修饰α-连环蛋白 力响应性；2)αE-连环蛋白构象和力传递特性如何受其影响 与它的其他连接伙伴结合。

The development and homeostasis of solid tissues depends upon biochemical and mechanical signals that control cell fate and the resulting organization of cells in the tissue. The conserved protein β-catenin is a key effector of signals from both the Wnt family of secreted growth factors that specify cell fate during embryogenesis and tissue renewal in the adult, and mechanical force transmitted through cell-cell junctions in multicellular tissues. We hypothesize that mechanical force transmitted through β-catenin links Wnt signaling and cell-cell adhesion, and our overall goal is to understand the molecular mechanisms underlying these dual roles of β-catenin. Our strategy is to use biochemical, structural and biophysical methods to address critical knowledge gaps in these areas. In the absence of Wnts, the β-catenin is bound in a “destruction complex” that includes the proteins Axin and Adenomatous Polyposis Coli (APC), and kinases that phosphorylate β-catenin; phosphorylation leads to ubiquitylation and destruction of β-catenin by the proteasome. Wnt binding to the receptors Frizzled (Fzd) and LRP5/6 enables Fzd to recruit the cytoplasmic protein Dishevelled (Dvl), which in turn binds to Axin and thereby recruits the destruction complex to the activated receptor complex. This leads to phosphorylation of the LRP5/6 intracellular domain, which inhibits β-catenin destruction; the stabilized β-catenin enters the nucleus and activates target genes. We will address critical mechanistic aspects of this pathway that are not understood: 1) how secreted ligands “activate” the Fzd-Dvl interaction needed for β-catenin stabilization through interaction with the extracellular cysteine-rich domain of Fzd and LRP5/6; 2) how activated Dvl recruits Axin to turn off β-catenin destruction; 3) how the β-catenin destruction complex forms and interacts with the ubiquitylation/proteosomal machinery; 4) the essential role of APC in β-catenin destruction. Force transmission through cell-cell adherens junctions (AJ) requires a complex of E-cadherin, β-catenin, and α-catenin, which binds to actin filaments and forms a minimal force-sensing unit. Tension on cadherins can release β-catenin and cause its translocation to the nucleus independent of, but synergized by, Wnt signaling. Understanding such tension-triggered release of β-catenin requires understanding how force is transmitted through the AJ complex. α-Catenin additionally has a central role in organizing epithelial tissues based on its interactions with vinculin, Epithelial Protein Lost in Neoplasm (EPLIN), the tight junction (TJ) protein Zonula Occludens (ZO)-1, and afadin, all of which bind actin and recruit other scaffolding and signaling proteins. We will study: 1) The force-dependent conformational landscape and force responsivness of α-catenin alone and bound to its partners, including how β-catenin modifies α-catenin force responsiveness; 2) How αE-catenin conformation and force transmission properties are affected by its binding to its other junctional partners.