Integrin Recycling and Adhesion Formation in Cell Migration

摘要 整合素是几乎所有细胞用于迁移的主要细胞黏附分子。在粘连过程中，整合素 聚集形成各种复合体，包括新生的粘连、小的灶状复合体和成熟的灶状粘连。 通过这些部位，细胞对底物施加张力。关于粘附力的组装，我们知道的很多 复合体来自对细胞在细胞外基质(ECM)配体上分离和复制的研究。这个细胞正在扩散 化验对于理解粘连形成的机制是强有力的，但它并不能完全概括关键特征。 以及细胞迁移过程中的黏附形成行为。特别是，它没有解释极化形成的原因 而没有考虑到整合素的内吞循环，这是至关重要的 用于在2D和3D环境中进行细胞迁移。我们已经开发出一种新的焦点黏附组装分析方法，即 严重依赖于粘连中整合素的内吞作用所产生的可循环整合素。有趣的是，这种整合素 以未连接的活性构象运行，并导致在前导形成高度极化的粘连 迁移细胞的边缘。我们将使用细胞生物学、生化和高分辨率成像方法来测试 假设回收的整合素在靠近前沿的胞吐部位附近播撒新的黏附形成。 我们的三个目标是：1)研究整合素循环、激活状态、胞外位置与黏附的关系 形成，2)确定微管细胞骨架如何有助于粘连的极化形成 回收的整合素和，3)确定细胞外基质的拓扑结构和硬度如何影响黏附形成 回收的整合素。在整个过程中，我们将检查回收的整合素形成的粘连如何影响能力 要迁移的细胞的数量。这项工作将促进对细胞用来极化其粘连的基本机制的理解 细胞迁移，这可能导致在细胞迁移不受控制的情况下进行干预的新策略，例如 癌症转移和持续性炎症。

Summary Integrins are the major cell adhesion molecules used by virtually all cells to migrate. During adhesion, integrins are clustered to form a variety of complexes including nascent adhesions, small focal complexes and mature focal adhesions. Through these sites, the cell exerts tension on the substratum. Much of what we know about the assembly of adhesion complexes comes from studies on cells detached and replated on extracellular matrix (ECM) ligands. This cell spreading assay has been powerful for understand mechanisms of adhesion formation, yet it does not fully recapitulate key features and behaviors of adhesion formation during cell migration. In particular, it does not account for the polarized formation of adhesions near the front of the cell and does not take into account the endocytic recycling of integrins, which is critical for cell migration in both 2D and 3D environments. We have developed a new focal adhesion assembly assay that is critically dependent on recycled integrin derived from the endocytosis of integrins in adhesions. Interestingly, this integrin travels in an unliganded and active conformation and leads to the highly polarized formation of adhesions at the leading edge of migrating cells. We will use cell biological, biochemical and high resolution imaging approaches to test the hypothesis that recycled integrin acts to seed new adhesion formation near sites of its exocytosis near the leading edge. Our three aims are: 1) to examine the relationship between integrin recycling, activation state, exocytic sites and adhesion formation, 2) to determine how the microtubule cytoskeleton contributes to the polarized formation of adhesions from recycled integrins and, 3) to determine how extracellular matrix topology and stiffness affect adhesion formation from recycled integrins. Throughout, we will examine how the formation of adhesions from recycled integrins affect the ability of cells to migrate. This work will advance understanding of the basic mechanisms cells use to polarize their adhesions for cell migration, which may lead to new strategies for intervention in cases where cell migration is unregulated, such as cancer metastasis and persistent inflammation.