Molecular and Force Dynamics: Leukocyte Adhesion Molecules

描述（由申请方提供）：白细胞向急性炎症部位的募集是一个精心策划的过程，由白细胞和内皮细胞粘附分子（CAM）（包括选择素、整联蛋白和Ig超家族配体）的膜表达和功能活化启动。在R 01的任期内出现了一系列统一的主题，这些主题提供了对PMNs如何整合力作为从滚动到牢固停滞和跨内皮迁移的过渡中的空间机械线索的分子尺度洞察：1）选择素被赋予机械和生物化学性质，这使得它们能够充当粘附和信号转导受体; 2）剪切应力和跨膜钙释放激活的Ca 2+（CRAC）通道调节细胞内钙流，其功能是同步整合素介导的停滞和细胞迁移; 3）22-整合素亲和力和结合ICAM-1的键机制通过调节跨内皮迁移的外向内信号提供了一种看门人机制。在这次竞争性更新中，我们将我们创新的血管模拟微流体通道与实时免疫荧光成像相结合，以实现以下特定目标：1）检查钙释放激活的钙流量在中性粒细胞募集的多步骤过程中的作用。2)确定中性粒细胞上的E-选择素配体如何作为机械换能器，触发和放大炎症内皮细胞上整合素活化的趋化因子信号传导3）定义中性粒细胞形状极化的LFA-1外向内信号传导的分子和机械机制。我们的策略需要使用新鲜分离的人中性粒细胞和小鼠炎症模型，其总体目标是确定炎症疾病预后和治疗的调控途径和分子靶点。

DESCRIPTION (provided by applicant): Recruitment of leukocytes to sites of acute inflammation is a finely orchestrated process initiated by membrane expression and functional activation of leukocyte and endothelial cell adhesion molecules (CAMs) including selectins, integrins, and Ig-super family ligands. A set of unifying themes have emerged over the tenure of this R01 which provide molecular scale insight into how PMNs integrate force as a spatio-mechanical cue in the transition from rolling to firm arrest and transendothelial migration: 1) Selectins are endowed with mechanical and biochemical properties which allow them to function as both adhesive and signal transduction receptors; 2) Shear stress and transmembrane calcium release-activated Ca2+ (CRAC) channels regulate intracellular calcium flux which functions to synchronize integrin mediated arrest and cell migration; 3) 22-integrin affinity and bond mechanics in binding ICAM-1 provide a gatekeeper mechanism via regulation of outside-in signaling of transendothelial migration. In this competitive renewal we apply our innovative vascular mimetic microfluidic channels combined with real-time immunofluorescence imaging to pursue the following specific aims: 1) Examine the role of calcium release activated calcium flux in orchestration of the multistep process of neutrophil recruitment. 2) Determine how E-selectin ligands on neutrophils serve as mechano-transducers that trigger and amplify chemokine signaling of integrin activation on inflamed endothelium 3) define the molecular and mechanical mechanisms underlying LFA-1 outside-in signaling of neutrophil shape polarization. Our strategy entails the use of freshly isolated human neutrophils and murine models of inflammation with the overarching goal of identifying regulatory pathways and molecular targets for prognosis and treatment of inflammatory diseases.