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: 10456097
• 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/10456097
• 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; mass spectrometric imaging; 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.

项目摘要 细胞外囊泡（EV）是膜包裹的结构，其中包含蛋白质，RNA，脂质和 从大多数（如果不是全部）释放的代谢产物介导细胞间通信。电动汽车的角色 在生理过程以及包括神经退行性疾病和癌症在内的病理状况 已建立。考虑到各种体液中的电动汽车的存在，也有极大的兴趣使用 这些囊泡是用于治疗剂的疾病检测和工程电动汽车的生物标志物。 研究模型系统中已识别细胞的EV释放，该电动汽车包含荧光标记的货物 秀丽隐杆线虫可以提供有关未解决的有关EV生物发生机制的未解决问题 和体内货物选择。我们发现钙稳态调节器离子通道CLHM-1是货物 在从男性特异性感觉神经元纤毛释放的电动汽车中。值得注意的是，当我们共同表达tdtomato- 用GFP标记的PKD-2标记为CLHM-1，这是一种已知的EV货物蛋白，在同一神经元中表达，我们很少 观察到囊泡中荧光蛋白的共定位，这表明CLHM-1和PKD-2在不同 EV亚群。我们发现含有电动汽车的PKD-2和CLHM-1不使用相同 生物发生和释放机制以不同的量排出，并且没有相同的 生理功能。我们的总体目标是借鉴我们的遗传系统的优势并切割 边缘成像和质谱方法来定义EV形成的机制 EV异质性的亚群和生理意义。 我们拟议的研究将利用我们独特的转基因动物，这些动物表达荧光标记为电动汽车的货物 在内源水平。高级成像技术，包括与飞行器检测的共聚焦显微镜和 用于传输电子显微镜的免疫金标签将使我们能够表征大小，形态，形态， 电动汽车的纤毛释放位点以及纤毛膜中侧脂不对称对货物的影响 排序。通过候选方法，我们将定义Flippases，floppase和Scramblases的作用，这些作用 控制EV子集的生物发生中的跨贝贝脂质不对称性。然后，我们将探讨细胞应力 破坏质膜磷脂稳态的条件会影响EV货物分类和释放。到 确定CLHM-1 EV子集中的其他货物，我们将在GFP标记的CLHM-1上执行质谱法 通过流式细胞仪分离的囊泡。最后，我们将确定雌雄同体衍生的刺激 含有男性纤毛神经元的EV的CLHM-1形成以及EV的重要性 释放动物交流和睫状功能。这项工作将导致人们了解 单个细胞产生具有不同生理功能的异质EV群体，从而广泛影响 关于我们对体内利用的基本生物发生和货物分选机制的理解。