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.

项目摘要 分泌的胞外小泡(EV)在细胞间的通讯中起着至关重要的作用 生理和病理过程。初级纤毛是一种从细胞中突出的感觉细胞器， 作为通过释放微泡来传递信号的平台，微泡是一类直接从血浆中萌发的电动汽车 薄膜。在线虫中，从感觉神经元纤毛脱落的微泡被释放到环境中。 它们在动物与动物之间的交流中发挥作用。我们发现CLHM-1，它的同源基因 照亮可药物基因组(IDG)靶FAM26离子通道，是从睫毛释放的电动汽车中的货物 基地。TD番茄标记的CLHM-1和GFP标记的PKD-2，一个很好描述的电动汽车货物和IDG的同源物 靶向PKD2L2 TRPP通道，共定位于睫状基和纤毛中段。然而，这些蛋白质 在不同数量的EV亚群中富含，仅PKD-2位于 在纤毛远端末端，EVS从这个部位脱落。含CLHM-1和PKD-2 EVS的差异脱落 进入环境对交配伙伴的存在作出反应，表明单个纤毛可以 释放不同的微泡亚群，每个亚群具有不同的货物浓缩和功能。 目前对微囊货物装载、生物发生和运输的机制了解有限。 放手。膜脂磷脂酰肌醇4，5-二磷酸PI(4，5)P2在蛋白质中起关键作用 定位和细胞骨架重塑。虽然一些研究表明PI(4，5)P2水平会影响电动汽车货物 关于PI(4，5)P2的定位和生物发生，目前还不清楚是促进还是抑制微泡的脱落。 PI(4，5)P2主要由I型磷脂酰肌醇4-磷酸5-激酶(PIP5K1s)，IDG 这项提案的目标重点。线虫中只有一个PIP5K1，即PPK-1。我们的首要目标是抽签 基于我们的遗传模型系统和尖端成像方法的优势，我们可以定义PIP5K1- PI(4，5)P2的依赖生成影响电动汽车的货物分类和编队。 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作为 该激酶的可诱导性丢失会影响纤毛来源的微泡亚群的脱落和 电动汽车中的货物代管。使用由PLCδ1的PH结构域组成的PI(4，5)P2报告 我们将确定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睫下定位是EV亚群丰度和 释放，以回应交配伴侣的存在。这项工作将导致对PIP5K1活动的理解 影响具有不同生理功能的异质EV种群的形成，对 我们对EV的生物发生和体内利用的货物分拣机制的理解。