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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.

项目摘要白细胞整合素的调节和分选是细胞黏附和极性的基本问题对各种炎症性疾病和自身免疫性疾病有很大的意义，但与成纤维细胞中的细胞黏附受体。我们建议研究白细胞整合素的分选以及在免疫细胞黏附和迁移中发挥关键作用的Tetraspanin蛋白。我们建议采用其动态分选依赖于一种新的F杆依赖机构，该机构不仅依赖于曲率，而是在一个特定范围的浅曲率上。我们还建议，这个机制正在管理中尺度模式这些受体被组装，包括传播梯度和皮质振荡，将贩运事件与细胞极性联系起来。具体地说，在目标1中，我们计划将包括整合素αMβ2在内的潜在跨膜蛋白的表面表达和动力学和CD81之后，系统地鉴定和验证了膜货物的内吞途径，由 F-bar蛋白FBP17和CIP4的非偏向蛋白质组学方法。在目标2中，我们的目标是分离该因子使用定义良好的体外系统测量膜的曲率和张力，并确定它们对F- 棒状膜的结合和微管形成。我们将使用基于纳米棒的支撑双层系统来关键评估F-bar蛋白是否感觉到弯曲。我们还将检验这样一种假设，即膜管形成可能需要脂质分选和主动膜力学。这些实验将提供一个定量的 F-bar蛋白如何在无疏水插入的情况下管状膜的生物物理理解其他BAR蛋白普遍使用的机制。在目标3中，我们将剖析通过检查自发移徙或监禁下的移徙来改变贩运情况。特别是，我们将探讨极化膜受体梯度形成的机制。总体而言，组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对白细胞整合素的调节和功能提供关键的见解以及在多个时空尺度上运行的监管机制，这些机制尚未调查过了。