Signal relay during directed cell migration

摘要 感知和传播驱动定向迁移的外部线索的属性是一个基本属性 是生物系统的重要组成部分，对包括胚胎发生在内的生理和病理过程至关重要， 成体组织稳态、炎症和免疫反应以及转移性侵袭。这项建议旨在 了解趋化性信号是如何在相邻细胞之间包装和传播的， 趋化性为此，我们研究了人类中性粒细胞，正常人血液中最丰富的白细胞。当 暴露于病原体分泌的主要化学引诱物，如N-甲酰基-Met-Leu-Phe（fMLF） 当中性粒细胞侵入机体并在损伤部位坏死时，中性粒细胞迅速发生极化， 以有效地向上迁移fMLF梯度。当中性粒细胞对fMLF反应时， 化学引诱物，其用于保持对初级化学引诱物信号的鲁棒性和敏感性。 我们确定了次级化学引诱物白三烯B4（LTB 4）是大规模招募所必需的。 中性粒细胞的损伤部位在体外和体内。为了让LTB 4成为真正的信号传递分子， 它必须以能够在趋化过程中产生稳定梯度的形式释放。在这一背景下， 我们确定LTB 4被包装在中性粒细胞的囊泡中，作为一种有效的传播途径， 相邻细胞之间的梯度。我们发现LTB_4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）， 化学引诱物刺激，释放其内容物作为外来体，从而作为包装机制， 传递趋化信号。此外，我们发现MVB的生物合成似乎是在核膜开始的 (NE)激活的中性粒细胞。我们假设NE是MVB形成的新位点， 将LTB 4合成机器包装成分泌性MVB，分泌性MVB释放外泌体以传递信号， 中性粒细胞趋化性为了验证这一假设，在目标1中，我们将直接可视化5-LO和FLAP动力学， 使用mCherry/GFP融合体和可光活化的报告基因在正常条件下和当 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FLAP突变体。由于积分 膜蛋白聚集被认为是有序膜微区的标志，在目标2中，我们将 确定核脂质微结构域在MVB生物发生中的作用。最后，在目标3中，我们将确定 通过评估ESCRT在这一过程中的作用， 加工和鉴定参与NE重塑的辅助蛋白。这个项目将提供很多 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的生物发生中的作用以及在MVB与NE之间的相互作用中带来前所未有的知识。 脂质和ESCRT依赖性途径之间的联系。

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.