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 In our unpublished results, we found that resolvin D1 (RvD1), a docosahexaenoic acid (DHA)–derived SPM, enhanced the acidification of the AC-containing compartments (efferosomes)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 随着细胞主动清除凋亡细胞(ACS)，细胞凋亡持续发生，称为 “胞吐作用”，以维持组织动态平衡。我们最近的研究揭示了泡泡细胞吞噬作用在 加速组织修复，因为它通过诱导专门的前体生物合成来促进炎症消退 阻止炎症反应的分解介质(SPM)。因此，了解泡沫化是如何发生的 成功地执行是至关重要的。关于AC的机制已经了解了很多 识别和摄取，但传出细胞如何降解ACS和处理代谢货物，如胆固醇 从AC消化中释放出来，是不完全理解的。此外，尽管吞噬作用和吞噬作用是相同的 共同的特征，如涉及细胞骨架重排和囊泡的细胞内运输 膜结合的货物，泡细胞是否劫持内吞机械来处理AC衍生的货物 目前仍不确定。在我们未发表的结果中，我们发现一种名为二十二碳六烯酸的分解蛋白D1(RvD1) (DHA)衍生的SPM，增强了含有AC的隔室(泡沫体)和LC3-II的酸化 脂化作用是LC3相关吞噬细胞(LAP)介导的身体降解的关键特征。正如我们最近的研究 表明Rvd1的生物合成需要激活泡饮受体MerTK，这些 结果表明，MerTK-RvD1信号在LAP介导的AC降解中具有新的作用。来研究是否有 关键的内吞调节因子-C末端Eps15同源结构域(EHD)蛋白，包括EHD1，EHD2， EHD3和EHD4-都参与了泡饮相关事件，我们分析了单细胞RNA测序 (scRNA-seq)来自动脉粥样硬化病变的数据集，其中许多细胞经历了凋亡，并发现EHD 巨噬细胞中有EHD1和EHD4高表达的蛋白质异质性表达 专业的泡沫状细胞。我们进一步发现，EHD1增加了细胞表面胆固醇流出的水平 巨噬细胞吞噬过程中转运蛋白ABCA1的表达，提示EHD1介导的内吞 ABCA1的转运可能在消除消化的急性冠脉综合征释放的过量游离胆固醇方面发挥作用。这里, 我们建议结合细胞生物学、生物化学、小鼠遗传学和功能基因组学的方法来 确定MerTK-RvD1信号在LAP(项目1)和EHD蛋白中的功能和机制 与胞吐相关的事件，包括维持细胞胆固醇稳态(项目2)和控制 MerTK的胞内转运(项目3)。所有这三个项目都需要共焦显微镜来成像AC- 在固定和活体中都含有隔室，包括泡小体、脂小体、内小体和溶酶体 细胞。鉴于我们对共焦显微镜的密集使用需求，本行政副刊要求 从徕卡微系统公司购买Stellaris 5共焦显微镜，这将使我们能够访问 补充信息层，以动态、结构性和机械性地洞察囊泡运输。在……里面 总而言之，Stellaris 5将显著加快我们在Parent R35获奖项目中所描述的研究。