Signal relay during directed cell migration

定向细胞迁移过程中的信号中继

• 批准号: 10436900
• 负责人: Phyllis I Hanson
• 金额: $ 53.68万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10436900
• 关键词: Actins; Actomyosin; Acute; Adopted; Adult; Affinity; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Architecture; Autoimmune; Automobile Driving; Back; Biochemical Genetics; Biogenesis; Biological; Blood; Blood Vessels; Caliber; Cell Nucleus; Cell membrane; Cells; Chemotactic Factors; Chemotaxis; Chronic Disease; Complex; Cues; Cytoskeleton; Disease; Electron Microscopy; Elements; Embryonic Development; Endocytosis; Enzymes; Exhibits; Exposure to; FPR1 gene; Generations; Golgi Apparatus; Health; Homeostasis; Human; Hydrolase; Immune response; In Vitro; Infection; Inflammation; Inflammatory; Injury; Innate Immune Response; Integral Membrane Protein; Invaded; Knowledge; LTB4R gene; Lamin Type A; Lead; Left; Leukocytes; Leukotriene A4; Leukotriene B4; Lipids; Membrane; Membrane Microdomains; Morphology; Multivesicular Body; Natural Immunity; Necrosis; Neutrophil Infiltration; Nuclear; Nuclear Envelope; Organelles; Pathologic Processes; Pathway interactions; Phospholipase A2; Physiological Processes; Process; Production; Property; Proteins; Reporter; Role; Signal Pathway; Signal Transduction; Site; Source; Sphingolipids; Testing; Tissues; Vesicle; biological systems; cell motility; constriction; driving force; exosome; genetic approach; immune health; in vivo; insight; lamin B receptor; lamin B2; lamin C; leucyl-phenylalanine; leucylmethionine; migration; mutant; neutrophil; novel; pathogen; recruit; response; tissue injury; trafficking

ABSTRACT The property of sensing and propagating external cues that drive directional migration is a fundamental property of biological systems, and is essential to physiological and pathological processes including embryogenesis, adult tissue homeostasis, inflammation and immune responses, and metastatic invasion. This proposal aims at understanding how chemotactic signals are packaged and propagated between neighboring cells during chemotaxis. To do so, we study human neutrophils, the most abundant leukocytes in normal human blood. When exposed to primary chemoattractants like N-formyl-Met-Leu-Phe (fMLF), which is secreted by pathogens invading the body and by necrotic cells at sites of injury, neutrophils rapidly undergo polarization that allows them to efficiently migrate up the fMLF gradient. As they react to fMLF, neutrophils secrete secondary chemoattractants that serve to maintain the robustness and sensitivity to the primary chemoattractant signals. We established that the secondary chemoattractant leukotriene B4 (LTB4) is required for the massive recruitment of neutrophils to sites of injury in vitro and in vivo. In order for LTB4 to act as a bona fide signal relay molecule, it must be released in a form that enables the generation of a stable gradient during chemotaxis. In this context, we established that LTB4 is packaged in vesicles in chemotaxing neutrophils as a way to effectively disseminate gradients between neighboring cells. We found that LTB4 and its synthesizing enzymes – 5-lipoxigenase (5-LO) and 5-LO activating protein (FLAP) - localize to intracellular multivesicular bodies (MBVs) which, upon chemoattractant stimulation, release their content as exosomes, thereby acting as a packaging mechanism to relay chemotactic signals. Further, we found that MVB biogenesis appears to be initiated at the nuclear envelope (NE) in activated neutrophils. We hypothesize that the NE is a novel site of MVB formation that enables packaging of the LTB4 synthetic machineryinto secretory MVBs that release exosomes to relay of signals during neutrophil chemotaxis. To test this hypothesis, in Aim 1 we will directly visualize 5-LO and FLAP dynamics in live cells using mCherry/GFP fusions and photoactivatable reporters under normal conditions and when endocytosis is blocked. We will also assess the role of FLAP clustering as a driving force for MVB biogenesis at the NE, by generating FLAP mutants with distinct affinities for the 5-LO substrate arachidonic acid. Since integral membrane proteins clustering is considered a hallmark of ordered membrane microdomains, in Aim 2 we will define the role of nuclear lipid micro-domains in MVB biogenesis. Finally, in Aim 3 we will establish the role of membrane remodeling complexes in the formation of the nuclear MVBs by assessing the role of ESCRTs in this process and identify accessory proteins involved in NE remodeling. This project is poised to provide much needed insight into the mechanisms regulating the genesis of chemotactic signals during neutrophil chemotaxis and will bring unprecedented knowledge into the role of the NE in the biogenesis of MVBs and in the interplay between lipid- and ESCRT-dependent pathways in their biogenesis.

抽象的 感知和传播驱动定向迁移的外部线索的特性是一个基本特性 生物系统的组成部分，对于包括胚胎发生在内的生理和病理过程至关重要， 成人组织稳态、炎症和免疫反应以及转移侵袭。该提案旨在 了解趋化信号如何在相邻细胞之间打包和传播 趋化性。为此，我们研究了人类中性粒细胞，这是正常人类血液中最丰富的白细胞。什么时候 暴露于病原体分泌的主要化学引诱剂，例如 N-甲酰基-Met-Leu-Phe (fMLF) 侵入身体并通过损伤部位的坏死细胞，中性粒细胞迅速发生极化，使它们能够 有效地向上迁移 fMLF 梯度。当中性粒细胞对 fMLF 做出反应时，它们会分泌次级 用于维持对主要化学引诱剂信号的鲁棒性和敏感性的化学引诱剂。 我们确定二次趋化剂白三烯 B4 (LTB4) 是大规模招募所必需的 中性粒细胞对体外和体内损伤部位的影响。为了让 LTB4 充当真正的信号中继分子， 它必须以能够在趋化过程中产生稳定梯度的形式释放。在此背景下， 我们确定 LTB4 被包装在趋化性中性粒细胞的囊泡中，作为有效传播的一种方式 相邻细胞之间的梯度。我们发现LTB4及其合成酶——5-脂氧合酶(5-LO) 和 5-LO 激活蛋白 (FLAP) - 定位于细胞内多泡体 (MBV)， 化学引诱剂刺激，以外泌体的形式释放其内容物，从而充当包装机制 传递趋化信号。此外，我们发现 MVB 生物发生似乎是在核膜处启动的 (NE) 存在于活化的中性粒细胞中。我们假设 NE 是 MVB 形成的新位点，使得 将 LTB4 合成机器包装成分泌性 MVB，释放外泌体以在过程中传递信号 中性粒细胞趋化性。为了检验这个假设，在目标 1 中，我们将直接可视化 5-LO 和 FLAP 动力学 在正常条件下和当 内吞作用被阻断。我们还将评估 FLAP 聚类作为 MVB 生物发生驱动力的作用 NE，通过生成对 5-LO 底物花生四烯酸具有不同亲和力的 FLAP 突变体。由于积分 膜蛋白聚集被认为是有序膜微域的标志，在目标 2 中我们将 定义核脂质微结构域在 MVB 生物发生中的作用。最后，在目标 3 中，我们将建立以下角色： 通过评估 ESCRT 在此过程中的作用，研究膜重塑复合物在核 MVB 形成中的作用 处理和鉴定参与 NE 重塑的辅助蛋白。该项目有望提供很多 需要深入了解中性粒细胞趋化过程中趋化信号发生的调节机制 并将为 NE 在 MVB 生物发生和相互作用中的作用带来前所未有的知识 生物发生中脂质依赖途径和 ESCRT 依赖途径之间的关系。