Impact of PIP5K1 on extracellular vesicle biogenesis

PIP5K1 对细胞外囊泡生物发生的影响

• 批准号: 10666794
• 负责人: Jessica E Tanis
• 金额: $ 15.58万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10666794
• 关键词: Affect; Afferent Neurons; Animal Communication; Animals; Biogenesis; Biological Models; Caenorhabditis elegans; Calcium; Categories; Cell membrane; Cells; Cilia; Communication; Comprehension; Cytoskeleton; Data; Distal; Endosomes; Environment; Eukaryotic Cell; Fluorescence Microscopy; Fostering; Generations; Genetic Models; Genome; Goals; Heterogeneity; Homeostasis; Homologous Gene; Human; Image; Imaging Techniques; Ion Channel; Knock-out; Lipid Bilayers; Lipids; Location; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Modeling; Neurodegenerative Disorders; Neurons; Organelles; PH Domain; PKD2 protein; Partner in relationship; Pathologic; Pathologic Processes; Pathway interactions; Phosphatidylinositols; Phosphotransferases; Physiological; Physiological Processes; Play; Population; Process; Proteins; Recycling; Reporter; Research; Research Personnel; Role; Sensory; Signal Transduction; Site; Sorting; Testing; Time; Transgenic Animals; Vesicle; Work; base; cell type; exosome; extracellular vesicles; genetic approach; genetic strain; imaging approach; in vivo; macromolecule; microvesicles; mutant; overexpression; phosphatidylinositol 4-phosphate; response; transmission process; tumor progression; variant of unknown significance; vesicular release

Project Summary Small secreted extracellular vesicles (EVs) play critical roles in communication between cells during physiological and pathological processes. The primary cilium, a sensory organelle protruding from cells, serves as a platform to transmit signals via shedding of microvesicles, a class of EVs that bud directly from the plasma membrane. In C. elegans, microvesicles shed from sensory neuron cilia are discharged into the environment where they play a role in animal-to-animal communication. We discovered that CLHM-1, the homolog of Illuminating the Druggable Genome (IDG) target FAM26 ion channels, is a cargo in EVs released from the ciliary base. tdTomato-tagged CLHM-1 and GFP-tagged PKD-2, a well described EV cargo and homolog of the IDG target PKD2L2 TRPP channel, colocalize in the ciliary base and cilium middle segment. However, these proteins are enriched in distinct EV subpopulations that are discharged in different quantities, with PKD-2 alone located in the cilium distal tip and EVs shed from this site. CLHM-1 versus PKD-2 containing EVs are differentially shed into the environment in response to the presence of mating partners, demonstrating that a single cilium can release distinct subpopulations of microvesicles, each with different cargo enrichment and functionalities. There is limited understanding of the mechanisms underlying microvesicle cargo loading, biogenesis and release. The membrane lipid phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 plays critical roles in protein localization and cytoskeletal remodeling. While a few studies suggest that PI(4,5)P2 levels affect EV cargo localization and biogenesis, it remains unclear whether PI(4,5)P2 promotes or inhibits microvesicle shedding. PI(4,5)P2 is predominantly generated by type I phosphatidylinositol 4-phosphate 5-kinases (PIP5K1s), the IDG target focus of this proposal. There is a single PIP5K1 in C. elegans, PPK-1. Our overarching goal is to draw upon the strengths of our genetic model system and cutting edge imaging approaches to define how PIP5K1- dependent generation of PI(4,5)P2 impacts EV cargo sorting and formation. The proposed research will use our unique transgenic animals that express multiple fluorescently tagged EV cargoes at single copy level and advanced imaging techniques to determine how overexpression of PPK-1 as well as inducible loss of this kinase affects shedding of cilia-derived microvesicle subpopulations and colocalization of cargoes in EVs. Using a a PI(4,5)P2 reporter consisting of the PH domain of PLCδ1 fused to GFP we will define the impact of PPK-1 on PI(4,5)P2 ciliary abundance and localization. Finally, we will determine if PPK-1 and PI(4,5)P2 subciliary localization underlies the differences in EV subpopulation abundance and release in response to mating partner presence. This work will lead to an understanding of how PIP5K1 activity impacts formation of heterogeneous EV populations with different physiological functions, impacting broadly on our comprehension of EV biogenesis and cargo sorting mechanisms utilized in vivo.

项目概要 小分泌型细胞外囊泡（EV）在细胞间的通讯中发挥着关键作用 初级纤毛是一种从细胞中伸出的感觉细胞器，发挥着生理和病理过程的作用。 作为通过微泡脱落来传输信号的平台，微泡是一类直接从血浆中萌芽的 EV 在秀丽隐杆线虫中，从感觉神经元纤毛脱落的微泡被排放到环境中。 我们发现了 CLHM-1，即 CLHM-1 的同源物。 照明可药物基因组 (IDG) 目标 FAM26 离子通道是从睫状体释放的 EV 中的货物 tdTomato 标记的 CLHM-1 和 GFP 标记的 PKD-2，一种详细描述的 EV 货物和 IDG 的同系物。 靶向 PKD2L2 TRPP 通道，共定位于纤毛基部和纤毛中段。 富含不同数量的不同 EV 亚群，其中仅 PKD-2 位于 在纤毛远端，从该位点脱落的 EV 与含有 PKD-2 的 EV 的脱落有差异。 进入环境以响应交配伙伴的存在，证明单个纤毛可以 释放不同的微泡亚群，每个亚群具有不同的货物富集和功能。 对微泡货物装载、生物发生和作用的机制了解有限。 膜脂磷脂酰肌醇 4,5-二磷酸 PI(4,5)P2 在蛋白质的释放中起着关键作用。 虽然一些研究表明 PI(4,5)P2 水平影响 EV 货物。 定位和生物发生，目前尚不清楚 PI(4,5)P2 是否促进或抑制微泡脱落。 PI(4,5)P2 主要由 I 型磷脂酰肌醇 4-磷酸 5-激酶 (PIP5K1s)（IDG）产生 该提案的目标重点是秀丽隐杆线虫中存在一个 PIP5K1，即 PPK-1。 凭借我们的遗传模型系统和尖端成像方法的优势来定义 PIP5K1- PI(4,5)P2 的依赖生成会影响 EV 货物的分类和形成。 拟议的研究将使用我们独特的转基因动物，这些动物表达多种荧光标记的 EV 单拷贝水平的货物和先进的成像技术来确定 PPK-1 的过度表达如何作为 该激酶的诱导性丢失会影响纤毛衍生的微泡亚群的脱落， 使用由 PLCδ1 的 PH 结构域组成的 PI(4,5)P2 报告基因在 EV 中的共定位。 GFP 我们将定义 PPK-1 对 PI(4,5)P2 纤毛丰度和定位的影响 最后，我们将确定。 如果 PPK-1 和 PI(4,5)P2 亚纤毛定位是 EV 亚群丰度差异的基础， 响应交配伙伴的存在而释放这项工作将有助于了解 PIP5K1 的活性。 影响具有不同生理功能的异质 EV 群体的形成，广泛影响 我们对 EV 生物发生和体内利用的货物分类机制的理解。