Imaging and Cell Culture

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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.