Integrin Trafficking to Focal Adhesions

摘要 细胞组装、粘附和动态感知细胞外基质 (ECM) 的能力对于 多细胞生命。整合素是异二聚体 αβ 跨膜粘附受体家族，结合特异性 ECM 配体通过其胞外域并允许对细胞粘附、迁移、 分化和生存。适当的整合素功能对于组织形态发生至关重要，并且受到干扰 癌症、皮肤病、肌肉骨骼、心血管和炎症疾病。整合素的主要位点 基质参与位于动态微米级信号平台中，称为粘着斑（FA）。整合素可以 进出 FA 的横向交换，以及进出细胞表面的交通，这种交通影响 细胞迁移、侵袭和癌症转移。然而，尽管它很重要，但对细胞的了解 整合素在何处以及如何经历胞吐和胞吞运输的精确机制水平 由于难以直接观察活细胞中的整合素胞吐作用，因此一直具有挑战性。对此作出回应 挑战 我们最近生成了“外标记”整合素，其中含有 pH 敏感荧光团 pHluorin 或 化学遗传 Halo 标签插入细胞外环。这些外标记整合素提供了第一个 整合素胞吐作用的直接观察表明，与最初的预期相反，整合素 胞吐作用发生在 FA 的一个子集上。借鉴我们在整合素 (Calderwood) 和活细胞方面的专业知识 膜运输成像（Toomre），这项多研究者 R01 提案旨在测试主要的新功能 由这些结果产生的假设。在目标 1 中，我们测试了整合素被选择性递送到的假设 在亚基特异性、ECM 调节的过程中生长 FA。在目标 2 中，我们测试整合素的假设 内吞作用发生在一组不同的 FA 上，这些 FA 正在翻转，内吞的整联蛋白被回收到 不断增长的FA。此外，我们还探讨了回收 FA 的分子机制。最后，在目标 3 中，我们 检验以下假设：整合素依赖性纤连蛋白 (FN) 胞吐作用也发生在 FA 生长过程中，而 FN 内吞作用发生在正在翻转的 FA 处。我们的实验方法结合了新颖的外标记 整合素和基质构建体，具有新的可裂解 Halo 染料和 pH 切换，以跟随外吞作用 活细胞，以测试关于整合素传递位置及其潜在机制的新假设。

ABSTRACT The ability of cells to assemble, adhere to and dynamically sense the extracellular matrix (ECM) is essential for multicellular life. Integrins, a family of heterodimeric αβ transmembrane adhesion receptors, bind specific ECM ligands via their ectodomains and permit bidirectional communication vital for cell adhesion, migration, differentiation, and survival. Proper integrin function is paramount for tissue morphogenesis, and is perturbed in cancer, skin disorders, musculoskeletal, cardiovascular and inflammatory diseases. A major site of integrin- matrix engagement is in dynamic micron-sized signaling platforms, called focal adhesions (FA). Integrins can laterally exchange into and out of FA, but also traffic to and from the cell surface and this trafficking influences cell migration, invasion and cancer metastasis. However, despite its importance, understanding at the cellular and mechanistic level of precisely where and how integrins undergo exocytic and endocytic traffic has been challenging due to difficulties directly visualizing integrin exo-endocytosis in live cells. In response to this challenge we recently generated `ecto-tagged' integrins containing the pH-sensitive fluorophore pHluorin or a chemical-genetic Halo-tag inserted into an extracellular loop. These ecto-tagged integrins provided the first direct views of integrin exocytosis and revealed that, contrary to initial expectations, integrin exocytosis occurs at a subset of FA. Drawing on our expertise in integrins (Calderwood) and live-cell imaging of membrane trafficking (Toomre), this multi-investigator R01 proposal seeks to test major new hypotheses arising from these results. In Aim 1 we test the hypothesis that integrins are selectively delivered to growing FA in a subunit-specific, ECM-regulated process. In Aim 2 we test the hypotheses that integrin endocytosis occurs at a distinct set of FA that are turning over and that endocytosed integrins are recycled to growing FA. Furthermore, we probe molecular mechanisms involved in recycling to FA. Finally, in Aim 3 we test the hypothesis that integrin-dependent fibronectin (FN) exocytosis also occurs at growing FA, while FN endocytosis occurs at FA that are turning over. Our experimental approaches combine novel ecto-tagged integrins and matrix constructs with new cleavable Halo dyes and pH-switching to follow exo-endocytosis in live cells so as to test new hypothesis about where integrin is delivered and the underlying mechanisms.