Signal relay during directed cell migration

定向细胞迁移过程中的信号中继

• 批准号: 10214472
• 负责人: Phyllis I Hanson
• 金额: $ 52.92万
• 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/10214472
• 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 敏感性和传播外部线索的特性，即推动方向迁移是一种基本财产 生物系统的生物系统，对于包括胚胎发生在内的物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性侵袭。该提议的目的是 了解如何在相邻细胞之间包装和传播趋化信号 趋化性。为此，我们研究人类中性粒细胞，这是正常人血液中最丰富的白细胞。什么时候 暴露于原发性趋化剂，例如N-甲基甲基 - leu-phe（FMLF），该剂是病原体分泌的 在损伤部位入侵身体和坏死细胞，中性粒细胞迅速经历极化 有效地迁移FMLF梯度。当它们对FMLF反应时，中性粒细胞秘密次要 用于维持对主要化学吸引剂信号的鲁棒性和敏感性的化学吸引力。 我们确定大规模招募需要二级趋化剂白细胞B4（LTB4） 嗜中性粒细胞到体外和体内受伤部位。为了使LTB4充当真正的信号中继分子， 它必须以一种在趋化过程中产生稳定梯度的形式释放。在这种情况下， 我们确定LTB4在趋化中性粒细胞中包装在蔬菜中，以有效传播 相邻细胞之间的梯度。我们发现LTB4及其合成酶 - 5-脂氧酶（5-lo） 和5 -lo激活蛋白（皮瓣） - 位于细胞内多细胞体（MBV），该物体在上面 化学吸引剂刺激，将其含量释放为外泌体，从而充当包装机制 继电器趋化信号。此外，我们发现MVB生物发生似乎是在核包膜上启动的 （NE）在活化的中性粒细胞中。我们假设NE是一个新颖的MVB形成场所，可以实现 LTB4合成Machineryinto秘书MVB的包装释放外泌体以在信号中传递 中性粒细胞趋化性。为了检验这一假设，在AIM 1中，我们将直接可视化5-LO和襟翼动力学 使用MCHERRY/GFP融合和光活化记者在正常条件下以及何时使用光活化记者的活细胞 内吞作用被阻断。我们还将评估皮瓣聚类作为MVB生物发生的驱动力的作用 NE，通过产生对5-lo底物花生四烯酸具有不同亲和力的瓣突变体。自积分以来 膜蛋白聚类被认为是有序膜微域的标志，在AIM 2中，我们将 定义核脂质微域在MVB生物发生中的作用。最后，在目标3中，我们将确定 通过评估ESCRT在此中的作用，膜在形成核MVB中的膜重塑络合物 过程并识别与NE重塑有关的辅助蛋白。这个项目被中毒以提供很多 需要深入了解中性粒细胞趋化过程中调查趋化信号起源的机制 并将将前所未有的知识带入NE在MVB的生物发生和相互作用中的作用 在其生物发生中脂质依赖性途径之间。