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

项目摘要 细胞外囊泡（EV）是含有蛋白质、RNA、脂质和脂质的膜包裹结构。 从大多数（如果不是所有）细胞类型释放的代谢物，以介导细胞间通讯。电动汽车的作用 在生理过程以及包括神经变性疾病和癌症的病理状况中 已经建立。鉴于EV在不同体液中的存在， 这些囊泡作为疾病检测的生物标志物和用于治疗的工程EV。 研究含有荧光标记的货物的EV从模型系统中鉴定的细胞的释放 C. elegans可以提供关于EV生物发生保守机制的未解决问题的见解 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， 在从男性特异性感觉神经元的纤毛释放的EV中。值得注意的是，当我们共表达tdTomato- 用GFP标记的PKD-2标记CLHM-1，PKD-2是一种在相同神经元中表达的已知EV货物蛋白，我们很少 观察到荧光蛋白在囊泡中的共定位，表明CLHM-1和PKD-2是不同的， EV亚群。我们已经发现，含有PKD-2和CLHM-1的EV不利用相同的酶。 生物发生和释放机制，以不同的数量排放，并且不具有相同的 生理功能我们的首要目标是利用我们的遗传系统和切割的优势， 用边缘成像和质谱方法确定EV形成的潜在机制 亚群和EV异质性的生理意义。 我们提议的研究将利用我们独特的转基因动物，表达荧光标记的EV货物 在内生水平上。先进的成像技术，包括带有Airyscan检测的共焦显微镜， 用于透射电子显微镜的免疫金标记将使我们能够表征纳米颗粒的大小，形态， 和EV的纤毛释放位点以及睫状膜中的侧向脂质不对称性对货物的影响 分类通过候选方法，我们将定义翻转酶、翻转酶和乱序酶的作用， 在EV亚群的生物发生中控制跨双层脂质不对称。然后我们将探索细胞压力 破坏质膜磷脂稳态的条件影响EV货物分选和释放。到 为了鉴定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，我们将确定两性衍生的刺激，诱导一个 来自雄性纤毛神经元的含有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。这项工作将导致理解如何 单个细胞产生具有不同生理功能的异质EV群体， 我们对基本生物发生和体内货物分选机制的理解。

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.