Integrin Trafficking to Focal Adhesions

整合素运输至局部粘连

• 批准号: 10330379
• 负责人: DAVID A CALDERWOOD
• 金额: $ 43.89万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-16 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10330379
• 关键词: Address; Adhesions; Age; Autoimmune Diseases; Binding; Biochemistry; Biological Assay; Cardiovascular Diseases; Cardiovascular system; Cell Adhesion; Cell Shape; Cell surface; Cell-Matrix Junction; Cells; Chemical Models; Communication; Complex; Data; Development; Disease; Dyes; Endocytosis; Environment; Enzymes; Epidermolysis Bullosa; Exocytosis; Extracellular Matrix; Family; Fibronectins; Focal Adhesions; Functional disorder; Genetic; Growth; Image; Imaging Device; Inflammatory; Integrin Binding; Integrin alpha2; Integrin beta Chains; Integrins; Label; Life; Ligands; Malignant Neoplasms; Mediating; Membrane; Microscopy; Microtubules; Modeling; Molecular; Molecular Probes; Morphogenesis; Musculoskeletal; Neoplasm Metastasis; PHluorin; Pathway interactions; Physiologic pulse; Process; Recycling; Regulation; Relaxation; Research Personnel; Resolution; Role; Signal Transduction; Signaling Protein; Site; Source; Specificity; Supporting Cell; Syndrome; Testing; Tissues; adhesion receptor; cancer cell; cell motility; chemical genetics; expectation; extracellular; fluorophore; inhibitor; innovation; live cell imaging; migration; novel; optogenetics; receptor; receptor binding; response; skin disorder; tool; trafficking; wound healing

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.

抽象的 细胞组装，遵守并动态感知细胞外基质（ECM）的能力对于 多细胞生活。整联蛋白是异二聚体αβ跨膜粘合剂的家族，结合了特异性 ECM配体通过其胞外域，并允许双向通信对于细胞粘附，迁移， 分化和生存。适当的整联蛋白功能对于组织形态发生至关重要，并且受到干扰 在癌症中，皮肤疾病，肌肉骨骼，心血管和炎症性疾病。整联蛋白的主要网站 - 矩阵的接合在动态微米大小的信号平台中，称为焦点粘合剂（FA）。整合素可以 横向交换进出FA，但也从细胞表面往返于及其贩运的影响 细胞迁移，侵袭和癌症转移。但是，它的重要性，在细胞上的理解 和整合素的机械水平，整合素的何处以及如何经历外吞和内吞交通 由于难以直接可视化活细胞中整联蛋白外胞胞性细胞增多症而受到挑战。为此回应 挑战我们最近产生了含有pH敏感荧光团植物或A 化学遗传光环标签插入细胞外环。这些ecto标记的整合素提供了第一个 整联蛋白胞吐作用的直接视图，并揭示了与初始期望形成对比的整合素 胞吐作用发生在FA的子集中。利用我们在整合素（Calderwood）和Live-Cell方面的专业知识 膜贩运的成像（Toomre），该多入侵者R01提案旨在测试主要的新型新 这些结果引起的假设。在AIM 1中，我们检验了整联蛋白被选择性地传递到的假设 在亚基特异性的ECM调节过程中生长FA。在AIM 2中，我们测试了整合素的假设 内吞作用发生在一组不同的FA中，该FA转移，内吞整合素被回收为 成长的FA。此外，我们探测与回收到FA的分子机制。最后，在目标3中我们 检验以下假设，即在生长的FA时，依赖整联蛋白依赖蛋白依赖蛋白（FN）的胞毒性也发生 内吞作用发生在FA上，正在旋转。我们的实验方法结合了新颖的ecto标签 具有新的可裂解光环染料和pH开关的整合素和基质构建体遵循外胞吞作用 活细胞以检验有关整联蛋白在何处和基本机制的新假设。