Imaging and Cell Culture

摘要 该项目的重点是研究重新密封的分子和信号机制， 粘附连接（AJs）和肺内皮屏障的恢复以及炎症后肺内环境的稳定 损伤这些过程将通过蛋白质分布的动态变化来定义，包括 AJs，包括信号分子，评估其在板状伪足水平的动力学和活性 突起和AJs。核心B将为所有项目提供技术支持，以解决关键信号的作用 所提出的参与肺内皮屏障恢复的分子，包括血管内皮蛋白 酪氨酸磷酸酶（VE-PTP）在项目1中，6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶3（PFK- FB 3），项目3中的鞘氨醇-1-磷酸受体-1（S1 PR 1），和项目4中的磷脂酶D2（PLD 2）。 项目4。这将涉及先进的活细胞成像和最先进的图像分析，以研究（i） 粘附蛋白（如VE-钙粘蛋白）和信号蛋白在AJs和板状伪足中的分布和动力学; (ii)PFK-FB 3和PLD 2在片状伪足形成和动力学以及AJs重新密封中的关键作用;（iii） 小RhoA GTP酶Rac 1、Cdc 42和RhoA的时空活性;（iv）光活化探针的利用 以控制信号分子在特定位点如AJs的活性;（v）机械肌动蛋白-肌球蛋白张力 跨VE-钙粘蛋白粘附的研究。核心B中提供的这些方法将提供高级 解决所提出的问题所需的成像能力。此外，Core B将提供高质量的 人和小鼠肺微血管内皮细胞的均匀培养和肺内皮细胞的分离 根据需要从遗传小鼠模型中提取。成像和细胞培养核心B对于满足 每个项目和整个计划的科学目标。

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.