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

项目摘要 细胞外小泡(EV)是一种膜包裹的结构，含有蛋白质、RNA、脂类和 从大多数细胞类型(如果不是所有细胞类型)释放的代谢物，以调节细胞间的通讯。电动汽车的角色 在生理过程和病理条件下，包括神经退行性疾病和癌症 已经建立起来了。鉴于电动汽车存在于不同的体液中，人们也对使用 这些囊泡作为疾病检测和治疗工程电动汽车的生物标志物。 模型系统中含有荧光标记货物的电动汽车从已识别细胞中释放的研究 线虫可以洞察有关EV生物发生保守机制的悬而未决的问题 和活体内的货物选择。我们发现钙稳态调节剂离子通道CLHM-1为Cargo 在男性特有感觉神经元的纤毛释放的EVS中。值得注意的是，当我们共同表达tdTomato- 用GFP标记的PKD-2标记CLHM-1，这是一种在相同神经元中表达的已知EV货运蛋白，我们很少 在囊泡中观察到荧光蛋白的共存，表明CLHM-1和PKD-2是不同的 EV亚群。我们发现含有EVS的PKD-2和CLHM-1不利用相同的 生物发生和释放机制，有不同的排泄量，也没有相同的 生理功能。我们的首要目标是利用我们基因系统的优势和 边缘成像和质谱学方法确定EV形成的潜在机制 EV异质性的亚群及其生理意义。 我们提议的研究将利用我们独特的转基因动物来表达带有荧光标记的EV货物 在内生水平上。先进的成像技术，包括带有AiryScan检测的共焦显微镜和 免疫金标记法用于透射电子显微镜将使我们能够表征大小，形态， 和睫状肌释放部位(S)，以及睫状膜横向脂质不对称对货物的影响 分类。通过候选方法，我们将定义翻转酶、翻转酶和加扰酶的角色，其中 控制跨双分子层脂质不对称，在EV亚群的生物发生中。然后我们将探索细胞压力是如何 破坏质膜磷脂动态平衡的条件会影响EV货物的分类和释放。至 识别CLHM-1电动汽车亚群中的其他货物，我们将对GFP标记的CLHM-1进行质谱分析 用流式细胞仪分离囊泡。最后，我们将识别两性人衍生的刺激，它会诱导 含CLHM-1的雄性纤毛神经细胞形成EVS的增加及其重要性 用于动物交流和睫毛功能的释放。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，影响广泛 关于我们对基本的生物发生和体内使用的货物分拣机制的理解。

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.