Integrin Trafficking to Focal Adhesions

整合素运输至局部粘连

• 批准号: 9973391
• 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/9973391
• 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/antagonist; 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，遵守和动态感的能力，细胞外基质（ECM）对于 多细胞寿命。 ECM配体通过它们的外生符，并允许双向传播通信对于细胞粘附，迁移，迁移至关重要。 区分和生存素。 在癌症中，皮肤疾病，肌肉骨骼，心腔和炎症疾病。 矩阵Engamic微米大小的信号平台，称为焦点粘连（FA） 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，发明和癌症转移，尽管它的重要性 正是您的位置以及Indergo外生和内吞作用的机械水平和机械水平 由于困难直接可视化活细胞中整合异常外胞吞作用的困难，因此具有挑战性。 挑战我们最近产生了含有pH敏感荧光团植物或A 化学基因晕圈插入细胞外环。 整联蛋白胞吐作用的直接观点，并揭示了与初始期望相反的整合素 胞吞作用发生在FA的子集中。 膜贩运成像（tomre），该多入侵者R01提案旨在测试主要的新型新 AIM 1中产生的假设。 在AIM 2中生长FA，在AIM 2中。 内吞作用发生在一组不同的FA中，该FA转向，内吞整合蛋白被回收为 进一步，我们探测涉及回收为FA的分子机制。 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 内吞作用发生在我们的实验方法中。 带有可裂解光环染料和pH开关的整合素和基质构建体遵循外胞吞作用 活细胞以检验整联蛋白上的新假设和下层机制。