Imaging and Cell Culture

成像和细胞培养

基本信息

批准号：
9324305
负责人：
Yulia A Komarova
金额：
$ 24.48万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9324305
关键词：
6-Phosphofructo-2-kinase Actomyosin Address Adherens Junction Adhesions Biosensor Blood Vessels Cell Culture Techniques Cell Separation Cell-Cell Adhesion Development Elements Endothelial Cells Energy Transfer Filopodia Fluorescence Fructose-2,6-bisphosphatase Fusion Protein Expression Generations Genes Genetic Genetic Models Guanosine Triphosphate Phosphohydrolases Homeostasis Human Ii-Key Image Image Analysis Imaging Techniques Immunofluorescence Immunologic Inflammatory Injury Lung Lung Inflammation Mechanics Methods Microscopic Modeling Molecular Mus Physiological Principal Investigator Process Property Protein Analysis Protein Dynamics Recovery Role Services Signal Transduction Signaling Molecule Signaling Protein Sphingosine-1-Phosphate Receptor Staining method Stains Structure of parenchyma of lung Vascular Permeabilities base cadherin 5 cellular imaging imaging capabilities injured live cell imaging lung injury meetings monolayer mouse model new therapeutic target particle phospholipase D2 programs protein distribution protein expression recombinant adenovirus restoration seal spatiotemporal vascular endothelial protein tyrosine phosphatase

项目摘要

Abstract The focus of the Program Project is to investigate the molecular and signaling mechanisms of re-sealing of adherens junctions (AJs) and restoration of lung endothelial barrier and homeostasis post-inflammatory lung injury. These processes will be defined by means of dynamic changes in the distribution of proteins comprising AJs including signaling molecules, assessment of their dynamics and activity at the level of lamellipodia protrusions and AJs. Core B will provide technical support for all Projects in addressing the role of key signaling molecules involved in lung endothelial barrier restoration as proposed including the vascular endothelial protein tyrosine phosphatase (VE-PTP) in Project 1, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFK- FB3) in Project 2, sphingosine-1-phosphate receptor-1 (S1PR1) in Project 3, and phospholipase D2 (PLD2) in Project 4. This will involve advanced live-cell imaging and state-of-art image analysis to investigate (i) distribution and dynamics of adhesion (such as VE-cadherin) and signaling proteins at AJs and in lamellipodia; (ii) key role of PFK-FB3 and PLD2 in lamellipodia formation and dynamics and the re-sealing of AJs; (iii) spatiotemporal activity of small RhoA GTPases Rac1, Cdc42 and RhoA; (iv) utilization of photoactivated probes to control the activity of signaling molecules at specific loci such as AJs; (v) mechanical acto-myosin tension across VE-cadherin adhesion using biosensors. These methods available in Core B will provide the advanced imaging capabilities needed to address the questions posed. In addition, Core B will provide high-quality uniformly cultured human and mouse lung microvessel endothelial cells and isolation of lung endothelial cells from genetic mouse models as needed. The Imaging and Cell Culture Core B will be essential for meeting the scientific objectives of each Project and the Program as a whole.

抽象的该计划项目的重点是研究重新密封的分子和信号传导机制附着连接（AJS）和肺内皮屏障和稳态后炎症后肺的恢复受伤。这些过程将通过包括包含蛋白质的分布的动态变化来定义 AJ包括信号分子，评估其动力学和活性在层状水平上突起和AJ。核心B将为所有项目提供有关关键信号的作用的技术支持所提出的包括血管内皮蛋白（包括血管内皮蛋白）参与肺内皮屏障恢复的分子在项目1，6-磷酸果糖-2-激酶/果糖-2,6-磷酸酶3（PFK-- FB3）在项目2中，项目3中的鞘氨酸-1-磷酸受体1（S1PR1）和磷脂酶D2（PLD2）中项目4。这将涉及先进的活细胞成像和最先进的图像分析来调查（i）粘附的分布和动力学（例如VE-钙粘着蛋白）和在AJS和Lamellipodia中的信号蛋白；（ii）PFK-FB3和PLD2在薄片形成和动力学以及AJ的重新密封中的关键作用；（iii）小的RhoA GTPases Rac1，cdc42和Rhoa的时空活性；（iv）利用光活化探针控制特定基因座（例如AJ）的信号分子的活性；（v）机械肌动蛋白张力使用生物传感器穿越VE-钙粘着蛋白的粘附。核心B中可用的这些方法将提供高级要解决提出的问题所需的成像功能。此外，核心B将提供高质量的均匀培养的人和小鼠肺微血管内皮细胞以及肺内皮细胞的分离根据需要根据遗传小鼠模型。成像和细胞培养核心B对于满足每个项目的科学目标和整个计划。