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 小的分泌性细胞外囊泡（EV）在细胞间的通信中起关键作用， 生理和病理过程。初级纤毛是从细胞中突出的感觉细胞器， 作为一个平台，通过微泡的脱落来传输信号，微泡是一类直接从血浆中出芽的EV 膜的In C.感觉神经元纤毛脱落的微泡被释放到环境中 它们在动物间的交流中扮演着重要的角色。我们发现，CLHM-1， 照亮可药用基因组（IDG）靶FAM 26离子通道，是从纤毛释放的EV中的货物。 基地tdTomato标记的CLHM-1和GFP标记的PKD-2，一种充分描述的EV货物和IDG的同源物 靶向PKD 2L 2 TRPP通道，共定位于睫状体基部和纤毛中段。然而，这些蛋白质 在不同的EV亚群中富集，这些亚群以不同的量排出，单独的PKD-2位于 纤毛末端和EV从该部位脱落。CLHM-1与含有PKD-2的EV差异性脱落 进入环境，以应对交配伙伴的存在，表明一个单一的纤毛可以 释放微泡的不同亚群，每个亚群具有不同的货物富集和功能。 对微囊泡货物装载、生物发生和生物降解的潜在机制的理解有限。 release.膜脂磷脂酰肌醇4，5-二磷酸PI（4，5）P2在蛋白质合成中起重要作用 定位和细胞骨架重塑。虽然一些研究表明PI（4，5）P2水平影响EV货物 尽管Pl（4，5）P2在微囊泡的定位和生物发生中起重要作用，但仍不清楚Pl（4，5）P2是否促进或抑制微囊泡脱落。 PI（4，5）P2主要由I型磷脂酰肌醇4-磷酸5-激酶（PIP 5 K1 s）、IDG 这是该提案的重点。在C语言中只有一个PIP 5 K1。elegans，PPK-1.我们的首要目标是 基于我们的遗传模型系统和尖端成像方法的优势，来定义PIP 5 K1- PI（4，5）P2的依赖性产生影响EV货物分类和形成。 这项拟议中的研究将使用我们独特的转基因动物，表达多种荧光标记的EV 货物在单拷贝水平和先进的成像技术，以确定如何过度表达PPK-1作为 这种激酶的可诱导损失影响纤毛衍生的微泡亚群的脱落， 货物在电动汽车中的共定位。使用由PLCδ1的PH结构域与PLCδ1的PH结构域融合组成的PI（4，5）P2报告基因， 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纤毛下定位是EV亚群丰度差异的基础， 释放以响应交配伴侣的存在。这项工作将导致了解PIP 5 K1活动如何 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生物发生和体内使用的货物分选机制的理解。