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基本属性 对包括胚胎发生在内的生理和病理过程是必不可少的， 成人组织的动态平衡，炎症和免疫反应，以及转移的侵袭。这项建议旨在 了解趋化信号是如何在相邻细胞之间打包和传播的 趋化性。为了做到这一点，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 接触主要的趋化物质，如病原体分泌的N-甲酰-蛋氨酸-亮氨酸-苯丙氨酸(FMLF) 中性粒细胞侵入身体和损伤部位的坏死细胞，迅速经历极化，从而使它们 以高效地向上迁移FMLF梯度。当中性粒细胞对FMLF发生反应时，它们会第二次分泌 化学诱导剂，用于保持对主要化学诱导剂信号的稳健性和敏感性。 我们确定了次级趋化物质白三烯B4(LTB4)是大规模募集所必需的 中性粒细胞在体外和体内的损伤部位。为了使LTB4发挥真正的信号传递分子的作用， 它的释放形式必须能够在趋化过程中产生稳定的梯度。在这方面， 我们确定LTB4被包装在趋化中性粒细胞的囊泡中，作为一种有效的传播方式 相邻单元格之间的渐变。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)-定位于细胞内多泡小体(MBV)，一旦 化学诱导剂的刺激，释放它们的内容作为外体，从而作为包装机制 传递趋化信号。此外，我们发现MVB的生物发生似乎是在核膜上启动的 (Ne)在激活的中性粒细胞中。我们假设NE是MVB形成的一个新地点，它使 将LTB4合成机包装成分泌型MVB，释放外切体以传递信号 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和襟翼动力学 在正常条件下和何时使用mCherry/GFP融合和光活化记者的活细胞 内吞作用受阻。我们还将评估瓣状物聚集作为MVB生物发生的驱动力的作用 NE，通过产生与5-LO底物花生四烯酸具有明显亲和力的翻盖突变体。自积分以来 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 明确核脂微域在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在核MVB形成中的作用来评价膜重塑复合体的形成 加工和鉴定参与NE重塑的辅助蛋白。这个项目将提供更多 需要深入了解中性粒细胞趋化过程中趋化信号产生的调控机制 并将把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 在它们的生物发生中，脂质依赖和ESCRT依赖的途径之间。

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.