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Endocytic dynamics and surface emergent property of leukocyte Integrins

白细胞整合素的内吞动力学和表面突现特性

基本信息

批准号：
10716618
负责人：
Min Wu
金额：
$ 48.22万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10716618
关键词：
Amino Acids Autoimmune Diseases Behavior Binding Biophysics CD81 gene Cell Adhesion Cell Culture Techniques Cell Polarity Cell membrane Cells Clathrin Coupled Data Disease Drug Targeting Endocytosis Event Extracellular Domain FDA approved Fibroblasts Genes Genome Homeostasis Human Genome Hydrophobicity Immune In Vitro Individual Inflammatory Integral Membrane Protein Integrins Lead Leukocyte Trafficking Leukocytes Link Lipids Macrophage-1 Antigen Mechanics Mediating Membrane Membrane Potentials Membrane Protein Traffic Membrane Proteins Morphology PIK3CG gene Pathway interactions Pattern Periodicals Physiological Play Property Proteins Proteomics Reaction Regulation Research Role Sorting Stable Isotope Labeling Surface System TRIP10 gene Technology Testing Travel Tubular formation Up-Regulation Work adhesion receptor cell motility cellular imaging design effective therapy experimental study genome editing insight knock-down live cell imaging migration novel protein degradation receptor reconstitution trafficking

项目摘要

Project Summary Regulation and sorting of leukocyte integrins are fundamental questions in cell adhesion and polarity that has great implication for various inflammatory and autoimmune diseases, but it has been poorly studied compared to cell adhesion receptors in fibroblast cells. We propose to investigate the sorting of leukocyte integrins and tetraspanin proteins that play critical role in immune cell adhesion and migration. We propose its dynamic sorting depends on a novel F-BAR-dependent mechanism that not only depends on curvature, but on a specific range of shallow curvature. We also propose that this mechanism is governing the mesoscale pattern these receptors are assembled, including propagating gradients and cortical oscillations, which link trafficking events to cell polarity. Specifically, in Aim 1, we plan to characterize surface expression and dynamics of these potential transmembrane protein cargos including integrin αMβ2 and CD81 after systematically identify and validate membrane cargos for the endocytic pathway mediated by F-BAR protein FBP17 and CIP4 using nonbiased proteomic approach. In Aim 2, we aim to isolate the factor of membrane curvature and tension using well-defined in vitro systems and to determine their effects on F- BAR membrane binding and tubulation. We will employ a nanobar-based supported bilayer system to critically evaluate if F-BAR protein senses curvature. We will also test the hypothesis that membrane tubulation may require lipid sorting and active membrane mechanics. These experiments will provide a quantitative biophysical understanding of how F-BAR proteins tubulate membranes without the hydrophobic insertion mechanisms commonly used by other BAR proteins. In Aim 3, we will dissect the functional consequences of the altered trafficking by examining spontaneous migration or migration under confinement. In particular, we will investigate the mechanism for polarized membrane receptor gradient formation. Collectively, combining advanced single cell imaging, genome-editing, proteomics, and in vitro reconstitution, the proposed research will shed key insights into the regulation and function of leukocyte integrins through sophisticated coordination and regulatory mechanisms operating at multiple spatial and temporal scales that have not yet been investigated.

项目概要白细胞整合素的调节和分类是细胞粘附和极性的基本问题，对各种炎症和自身免疫性疾病具有重要意义，但与之相比，其研究却很少成纤维细胞中的细胞粘附受体。我们建议研究白细胞整合素的分类和四跨膜蛋白在免疫细胞粘附和迁移中发挥关键作用。我们建议其动态排序依赖于一种新颖的依赖于 F-BAR 的机制，该机制不仅取决于曲率，但在特定的浅曲率范围内。我们还建议该机制负责管理这些受体组装的中尺度模式，包括传播梯度和皮质振荡，将运输事件与细胞极性联系起来。具体来说，在目标 1 中，我们计划描述这些潜在跨膜蛋白货物（包括整合素 αMβ2）的表面表达和动力学和 CD81 后系统地识别和验证膜货物的内吞途径介导使用无偏蛋白质组学方法分析 F-BAR 蛋白 FBP17 和 CIP4。在目标 2 中，我们的目标是隔离因素使用明确的体外系统测量膜曲率和张力，并确定它们对 F- 的影响 BAR 膜结合和管化。我们将采用基于纳米棒的支撑双层系统来关键评估 F-BAR 蛋白是否感知曲率。我们还将检验膜管可能的假设需要脂质分选和活性膜力学。这些实验将提供定量的对 F-BAR 蛋白如何在没有疏水插入的情况下管状膜的生物物理学理解其他 BAR 蛋白常用的机制。在目标 3 中，我们将剖析以下功能的后果通过检查自发移徙或禁闭下的移徙来改变贩运活动。特别是，我们将研究极化膜受体梯度形成的机制。综合起来，结合先进的单细胞成像、基因组编辑、蛋白质组学和体外重建，拟议的研究将通过复杂的协调揭示白细胞整合素的调节和功能的关键见解以及在多个空间和时间尺度上运作的监管机制尚未得到落实调查了。