Integrin Trafficking to Focal Adhesions

摘要 细胞组装、粘附和动态感知细胞外基质（ECM）的能力对于细胞的增殖和分化是必不可少的。 多细胞生命整合素是异源二聚体αβ跨膜粘附受体家族， ECM配体通过它们的胞外域，并允许双向通信至关重要的细胞粘附，迁移， 分化和生存。适当的整合素功能对于组织形态发生是至关重要的， 癌症、皮肤病、肌肉骨骼、心血管和炎症疾病。整合素的一个主要位点- 基质接合是在动态的微米大小的信号平台中，称为粘着斑（FA）。整合素可以 横向交换进出FA，但也运输到和从细胞表面，这种运输影响 细胞迁移、侵袭和癌症转移。然而，尽管它的重要性，理解在细胞 整合素在何处以及如何进行胞吐和胞吞运输的精确机制水平， 由于难以直接可视化活细胞中的整合素外吞-内吞作用，因此具有挑战性。针对这一 我们最近产生了含有pH敏感荧光团pHluorin或pHluorin的“外标记的”整联蛋白。 插入细胞外环的化学遗传Halo标签。这些外标记的整合素提供了第一个 整合素胞吐作用的直接观点，并显示，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），这个多研究者R 01提案旨在测试主要的新的 从这些结果中产生的假设。在目的1中，我们检验了整合素选择性地递送至 在亚基特异性ECM调节的过程中生长FA。在目标2中，我们检验了整合素 内吞作用发生在一组不同的FA上，这些FA正在翻转，并且内吞的整合素被再循环， 成长FA此外，我们探测参与回收FA的分子机制。最后，在目标3中， 检验整合素依赖性纤连蛋白（FN）胞吐也发生在生长的FA，而FN 内吞作用发生在FA的翻转。我们的实验方法结合了联合收割机新型的外标记 整合素和基质构建体与新的可切割的Halo染料和pH转换，以遵循外切-内吞作用， 活细胞，以测试关于整合素在哪里传递和潜在机制的新假设。

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.