Integrin Trafficking to Focal Adhesions

摘要 细胞组装、黏附和动态感知细胞外基质(ECM)的能力对于 多细胞生命。整合素是一类异源二聚体αβ跨膜黏附受体家族，具有结合特异性 ECM配体通过其胞外结构域，允许对细胞黏附、迁移 分化和生存。适当的整合素功能对组织形态发生至关重要，并受到干扰 在癌症、皮肤病、肌肉骨骼、心血管疾病和炎症性疾病中。整合素的一个主要部位- 基质参与是在动态微米级的信号平台中进行的，称为焦点粘连(FA)。整合素可以 横向交换进出FA，但也有进出小区表面的流量，这种贩运影响 细胞迁移、侵袭和癌症转移。然而，尽管它很重要，在细胞上的理解 以及整合素在哪里以及如何通过胞外和胞内运输的确切机制水平 由于难以直接观察活细胞中整合素的外吞吞作用，这一研究一直具有挑战性。作为对此的回应 挑战我们最近产生了含有pH敏感荧光团PHluorin或a的‘ecto标记’整合素 插入到细胞外环中的化学遗传晕标签。这些胞外标记的整合素提供了第一个 直接观察整合素的胞吐作用，发现与最初的预期相反，整合素 胞吐作用发生在FA的一个子集。利用我们在整合素(Calderwood)和活细胞方面的专业知识 膜运输成像(Toomre)，这项多调查者R01提案旨在测试主要的新 根据这些结果提出的假设。在目标1中，我们测试了整合素被选择性地输送到 在亚单位特定的、ECM调控的过程中生长FA。在目标2中，我们检验了整合素的假设 内吞作用发生在一组不同的FA上，这些FA正在翻转，内吞的整合素被循环到 不断增长的FA。此外，我们还对FA回收的分子机制进行了探讨。最后，在目标3中，我们 假设整合素依赖的纤维连接蛋白(FN)胞吐也发生在生长的FA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ecto-tag 整合素和基质构建的新的可切割的Halo染料和pH切换跟随外吞作用 以检验关于整合素在哪里传递和潜在机制的新假说。

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.