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 In our unpublished results, we found that resolvin D1 (RvD1), a docosahexaenoic acid (DHA)–derived SPM, enhanced the acidification of the AC-containing compartments (efferosomes)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.

抽象的 细胞凋亡随着胞外细胞主动清除凋亡细胞（AC）而持续发生，称为 “胞吞作用”，维持组织稳态。我们最近的研究揭示了胞吞作用的新作用 加速组织修复，因为它通过诱导专门的亲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，了解胞吞作用是如何进行的 成功开展至关重要。人们对 AC 的机制已经了解很多 识别和摄取，但传细胞如何降解 AC 并处理代谢货物，例如胆固醇 从 AC 消化中释放出来的物质，目前尚不完全清楚。此外，虽然胞吞作用和内吞作用共享 共同特征，例如涉及细胞骨架重排和囊泡的细胞内运输 膜结合的货物，传热细胞是否劫持内吞机制来处理 AC 衍生的货物 仍不确定。在我们未发表的结果中，我们发现 resolvin D1 (RvD1)，一种二十二碳六烯酸 (DHA) 衍生的 SPM，增强了含有 AC 的室（efferosome）和 LC3-II 的酸化 脂化，LC3 相关吞噬作用 (LAP) 介导的尸体降解的关键特征。正如我们最近的研究 研究表明，RvD1 生物合成需要激活 MerTK（胞吞作用受体），这些 结果表明 MerTK-RvD1 信号在 LAP 介导的 AC 降解中发挥新作用。要研究是否 关键的内吞调节因子——C 端 Eps15 同源域 (EHD) 蛋白，包括 EHD1、EHD2、 EHD3 和 EHD4 — 参与胞吞作用相关事件，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，其中许多细胞发生凋亡，并发现 EHD 蛋白质具有异质性表达，巨噬细胞中 EHD1 和 EHD4 高表达， 专业的传热细胞。我们进一步发现EHD1增强了细胞表面胆固醇流出的水平 胞吞作用期间巨噬细胞中的转运蛋白 ABCA1，这表明 EHD1 介导的内吞作用 ABCA1 的运输可能在去除消化 AC 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 确定 LAP（项目 1）和 EHD 蛋白中 MerTK-RvD1 信号传导的功能和机制 胞吞作用相关事件，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）。所有三个项目都需要共焦显微镜来成像 AC- 包含固定和活体中的胞质体、LAPosomes、内涵体和溶酶体等隔室 细胞。鉴于我们对共聚焦显微镜的大量使用需求，本行政补充要求 从 Leica microsystems 购买 STELLARIS 5 共焦显微镜，这将使我们能够访问 补充信息层，以了解囊泡运输的动态、结构和机制。在 总之，STELLARIS 5 将显着加速我们在资助的母公司 R35 奖项中描述的研究。