Efferocytosis meets endocytosis

胞吞作用遇上内吞作用

• 批准号: 10795494
• 负责人: Bishuang Cai
• 金额: $ 24.99万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10795494
• 关键词: ATP binding cassette transporter 1; Acceleration; Administrative Supplement; Anabolism; Apoptosis; Apoptotic; Arterial Fatty Streak; Award; Binding; Biochemistry; C-terminal; Carrier Proteins; Cell surface; Cells; Cellular biology; Cholesterol; Cholesterol Homeostasis; Confocal Microscopy; Cytoskeleton; Data Set; Digestion; Docosahexaenoic Acids; Endocytosis; Endosomes; Event; Funding; Homeostasis; Image; Inflammation; Inflammatory Response; Intracellular Transport; Learning; Lysosomes; Macrophage; Maintenance; Mediating; Mediator; Membrane; Metabolism; Microscope; Parents; Phagocytosis; Process; Research; Resolution; Role; Signal Transduction; Tertiary Protein Structure; Tissues; Vesicle; functional genomics; insight; microsystems; mouse genetics; novel; receptor; single-cell RNA sequencing; tissue repair; trafficking; uptake; vesicle transport

Abstract Apoptosis happens continuously along with the active clearance of apoptotic cells (ACs) by efferocytes, termed “efferocytosis”, to maintain tissue homeostasis. Our recent studies revealed a novel role of efferocytosis in accelerating tissue repair as it promotes inflammation resolution by inducing the biosynthesis of specialized pro- resolving mediators (SPMs) that stop inflammatory responses. Therefore, understanding how efferocytosis is successfully carried out is of paramount importance. Much has been learned about the mechanisms of AC recognition and uptake, but how efferocytes degrade ACs and process the metabolic cargo, e.g., cholesterol released from AC digestion, is incompletely understood. Moreover, although efferocytosis and endocytosis share common features, such as involving cytoskeleton rearrangement and intracellular transport of vesicular membrane-bound cargoes, whether efferocytes hijack the endocytic machinery to process AC- derived cargo remains uncertain. In our unpublished results, we found that resolvin D1 (RvD1), a docosahexaenoic acid (DHA)–derived SPM, enhanced the acidification of the AC-containing compartments (efferosomes) and LC3-II lipidation, key features in LC3-associated phagocytosis (LAP)-mediated corpse degradation. As our recent study showed that the activation of MerTK, the efferocytosis receptor, was required for RvD1 biosynthesis, these results indicate a novel role of MerTK-RvD1 signaling in LAP-mediated AC degradation. To study whether the key endocytic regulators—the C-terminal Eps15 Homology Domain (EHD) proteins comprising EHD1, EHD2, EHD3, and EHD4—are involved in efferocytosis-related events, we analyzed a single-cell RNA-sequencing (scRNA-seq) dataset from atherosclerotic lesions where a lot of cells undergo apoptosis and found that EHD proteins had heterogeneous expression with high expression of EHD1 and EHD4 in macrophages, the professional efferocytes. We further found that EHD1 enhanced the cell surface levels of the cholesterol efflux transport protein ABCA1 in macrophages during efferocytosis, which indicates that EHD1- mediated endocytic trafficking of ABCA1 may play a role in removing the excess free cholesterol released from digested ACs. Here, we propose to combine approaches in cell biology, biochemistry, mouse genetics, and functional genomics to determine the function and mechanisms of MerTK-RvD1 signaling in LAP (Project 1) and EHD proteins in efferocytosis-related events including maintaining cellular cholesterol homeostasis (Project 2) and controlling endocytic trafficking of MerTK (Project 3). All the three projects require confocal microscopy to image AC- containing compartments including efferosomes, LAPosomes, endosomes and lysosomes in both fixed and live cells. Given our intensive usage needs for confocal microscopy, this Administrative Supplement requests the acquisition of the STELLARIS 5 confocal microscope from Leica microsystems, which will allow us to access to complementary layers of information for dynamic, structural, and mechanistic insights into vesicle trafficking. In summary, the STELLARIS 5 will significantly accelerate our research described in the funded parent R35 award.

摘要 细胞凋亡沿着凋亡细胞（AC）被巨噬细胞主动清除的过程， “红细胞增多症”，以维持组织稳态。我们最近的研究揭示了红细胞增多症在 加速组织修复，因为它通过诱导专门的前- 解决介质（SPM），停止炎症反应。因此，了解红细胞增多症 成功实施是至关重要的。关于AC的机制已经有了很多了解 识别和摄取，但是巨噬细胞如何降解AC和处理代谢货物，例如，胆固醇 从AC消化中释放出来，还没有完全理解。此外，虽然胞吞作用和内吞作用共享 共同的特点，如涉及细胞骨架重排和细胞内运输的囊泡， 膜结合的货物，无论是巨噬细胞劫持内吞机制处理AC衍生的货物 仍然不确定。在我们未发表的研究结果中，我们发现一种二十二碳六烯酸， （DHA）衍生的SPM，增强了含AC的隔室（脂质体）和LC 3-II的酸化 脂质化，LC 3相关吞噬作用（CYP）介导的尸体降解的关键特征。作为我们最近的研究 表明，MerTK的激活，红细胞增多症受体，是RvD 1生物合成所必需的，这些 结果表明MerTK-RvD 1信号传导在LAP介导的AC降解中的新作用。研究是否 关键的内吞调节因子-C-末端Eps 15同源结构域（EHD）蛋白，包括EHD 1，EHD 2， EHD 3和EHD 4-参与细胞凋亡相关事件，我们分析了单细胞RNA测序， 在来自动脉粥样硬化病变的scRNA-seq数据集中，许多细胞经历凋亡，并发现EHD EHD 1和EHD 4在巨噬细胞中呈高表达， 专业的细胞。我们进一步发现，EHD 1增强细胞表面水平的胆固醇流出， 在巨噬细胞吞噬过程中转运蛋白ABCA 1，这表明EHD 1介导的内吞 ABCA 1的运输可能在去除从消化的AC释放的过量游离胆固醇中起作用。在这里， 我们建议将细胞生物学、生物化学、小鼠遗传学和功能基因组学的方法联合收割机， 确定MerTK-RvD 1信号转导在EHD蛋白（项目1）和EHD蛋白中的功能和机制， 细胞凋亡相关事件，包括维持细胞胆固醇稳态（项目2）和控制 MerTK的内吞运输（项目3）。所有这三个项目都需要共焦显微镜来成像AC- 在固定的和活的细胞中含有包括溶酶体、LAPosome、内体和溶酶体的隔室 细胞鉴于我们对共聚焦显微镜的密集使用需求，本行政补充要求 从徕卡显微系统公司购买STELLARIS 5共聚焦显微镜，这将使我们能够访问 互补的信息层的动态，结构和机械的见解囊泡贩运。在 总之，STELLARIS 5将大大加速我们在资助的父R35奖中描述的研究。