Elucidating biogenesis and cargo sorting mechanisms for discrete extracellular vesicle subpopulations in C. elegans

阐明线虫离散细胞外囊泡亚群的生物发生和货物分选机制

• 批准号: 10223381
• 负责人: Jessica E Tanis
• 金额: $ 32.91万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10223381
• 关键词: Afferent Neurons; Animal Behavior; Animal Communication; Animals; Biogenesis; Biological Markers; Biological Models; Body Fluids; Caenorhabditis elegans; Calcium; Candidate Disease Gene; Cell membrane; Cells; Cellular Stress; Chimeric Proteins; Cilia; Communication; Comprehension; Confocal Microscopy; Cultured Cells; Data; Defect; Detection; Development; Disease; Endocytosis; Engineering; Environment; Exclusion; Flow Cytometry; Genetic; Goals; Hermaphroditism; Heterogeneity; Homeostasis; Image; Imaging Techniques; Individual; Investigation; Ion Channel; Kinesin; Label; Lateral; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Membrane; Microscopy; Modeling; Molecular; Morphology; Multivesicular Body; Neurodegenerative Disorders; Neurons; Pathologic; Phospholipids; Physiological; Physiological Processes; Plasma; Play; Population; Population Heterogeneity; Proteins; RNA; Research; Resolution; Role; Site; Sorting - Cell Movement; Stimulus; Structure; System; Techniques; Testing; Therapeutic; Time; Tissues; Transgenic Animals; Transmission Electron Microscopy; Vesicle; Visualization; Work; animal imaging; base; cell type; exosome; extracellular vesicles; genetic approach; in vivo; insight; intercellular communication; interest; macromolecule; male; microvesicles; mutant; novel; prevent; tumor progression; vesicular release

Project Summary Extracellular vesicles (EVs) are membrane-wrapped structures containing proteins, RNAs, lipids, and metabolites that are released from most if not all cell types to mediate intercellular communication. Roles for EVs in physiological processes as well as pathological conditions including neurodegenerative diseases and cancer have been established. Given the presence of EVs in diverse body fluids, there is also great interest in using these vesicles as biomarkers for disease detection and engineering EVs for therapeutics. Investigation of the release of EVs containing fluorescently-tagged cargo from identified cells in the model system C. elegans can provide insight into unresolved questions concerning conserved mechanisms of EV biogenesis and cargo selection in vivo. We discovered that the calcium homeostasis modulator ion channel CLHM-1 is cargo in EVs released from cilia of male-specific sensory neurons. Remarkably, when we coexpressed tdTomato- tagged CLHM-1 with GFP-tagged PKD-2, a known EV cargo protein expressed in the same neurons, we rarely observed colocalization of the fluorescent proteins in vesicles, suggesting that CLHM-1 and PKD-2 are in distinct EV subpopulations. We have found that the PKD-2 and CLHM-1 containing EVs do not utilize the same biogenesis and release mechanisms, are discharged in different quantities, and do not have the same physiological function. Our overarching goal is to draw upon the strengths of our genetic system and cutting edge imaging and mass spectrometry approaches to define mechanisms underlying formation of EV subpopulations and the physiological significance of EV heterogeneity. Our proposed research will utilize our unique transgenic animals that express fluorescently tagged EV cargoes at endogenous levels. Advanced imaging techniques including confocal microscopy with Airyscan detection and immunogold labeling for transmission electron microscopy will enable us to characterize the size, morphology, and ciliary release site(s) of EVs as well as the impact of lateral lipid asymmetry in the ciliary membrane on cargo sorting. Through a candidate approach, we will define the role of flippases, floppases and scramblases, which control transbilayer lipid asymmetry, in the biogenesis of the EV subsets. We will then explore how cellular stress conditions that disrupt plasma membrane phospholipid homeostasis impact EV cargo sorting and release. To identify other cargoes in the CLHM-1 EV subset, we will perform mass spectrometry on GFP-tagged CLHM-1 vesicles isolated by flow cytometry. Finally, we will identify the hermaphrodite-derived stimulus that induces an increase in formation of CLHM-1 containing EVs from male ciliated neurons as well as the importance of EV release for animal communication and ciliary function. This work will lead to an understanding of how an individual cell generates heterogeneous EV populations with different physiological functions, impacting broadly on our comprehension of basic biogenesis and cargo sorting mechanisms utilized in vivo.

项目概要 细胞外囊泡 (EV) 是膜包裹的结构，含有蛋白质、RNA、脂质和 大多数（如果不是全部）细胞类型释放的代谢物，用于介导细胞间通讯。电动汽车的角色 生理过程以及病理状况，包括神经退行性疾病和癌症 已成立。鉴于电动汽车存在于不同的体液中，人们对使用电动汽车也产生了浓厚的兴趣 这些囊泡作为疾病检测的生物标志物和用于治疗的工程化 EV。 研究模型系统中已识别细胞释放含有荧光标记货物的 EV 线虫可以深入了解有关 EV 生物发生的保守机制的未解决问题 和体内货物选择。我们发现钙稳态调节剂离子通道 CLHM-1 是货物 从男性特异性感觉神经元纤毛释放的 EV 中。值得注意的是，当我们共表达 tdTomato- 标记 CLHM-1 和 GFP 标记的 PKD-2（一种在相同神经元中表达的已知 EV 货物蛋白），我们很少 观察到囊泡中荧光蛋白的共定位，表明 CLHM-1 和 PKD-2 处于不同的位置 EV 亚群。我们发现包含 EV 的 PKD-2 和 CLHM-1 不利用相同的 生物发生和释放机制，以不同的量排出，并且不具有相同的 生理功能。我们的首要目标是利用我们遗传系统的优势并削减 边缘成像和质谱方法来定义 EV 形成的机制 亚群和 EV 异质性的生理意义。 我们提出的研究将利用我们独特的转基因动物来表达荧光标记的 EV 货物 在内源水平上。先进的成像技术，包括具有 Airyscan 检测功能的共焦显微镜和 用于透射电子显微镜的免疫金标记将使我们能够表征尺寸、形态、 EV 和纤毛释放位点以及纤毛膜横向脂质不对称对货物的影响 排序。通过候选方法，我们将定义翻转、翻转和扰乱的作用，这 在 EV 子集的生物发生中控制跨双层脂质不对称性。然后我们将探讨细胞压力如何 破坏质膜磷脂稳态的条件会影响电动汽车货物的分类和释放。到 为了识别 CLHM-1 EV 子集中的其他货物，我们将对 GFP 标记的 CLHM-1 进行质谱分析 通过流式细胞术分离囊泡。最后，我们将确定引起雌雄同体的刺激 雄性纤毛神经元含有 CLHM-1 的 EV 的形成增加以及 EV 的重要性 释放用于动物交流和纤毛功能。这项工作将导致人们了解如何 单个细胞产生具有不同生理功能的异质 EV 群体，影响广泛 基于我们对体内使用的基本生物发生和货物分类机制的理解。