EAGER: Mechanistic Study of Extracellular Vesicle Production by Marine Microalgae using Advanced Imaging Technologies

EAGER：利用先进成像技术研究海洋微藻产生细胞外囊泡的机制

• 批准号: 2202723
• 负责人: Gordon Taylor
• 金额: $ 29.45万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2202723&HistoricalAwards=false
• 关键词: EAGER; Mechanistic; Study; Extracellular; Vesicle

This EAGER project is a proof-of-concept study on the composition, origins, and dynamics of extracellular vesicles (EVs) produced by marine microalgae in response to various environmental and biotic stressors. EVs are microscopic lipid-encased particles that are released naturally from almost all cell types and are vehicles for a variety of cargo, including genetic material (RNA, DNA), proteins, and lipids. EVs have been variously postulated to serve as a defense against viral attack, a waste disposal mechanism, a stress response, or a means of cell-to-cell communication. Marine microalgae are pivotal players in the global carbon cycle. By better understanding processes that govern their population dynamics and responses to environmental changes, we can develop better predictive models of responses to global climate change. The need to understand these mechanisms is becoming increasingly urgent as climate change becomes more manifest. Very recent findings suggest that EVs play a key role in marine phytoplankton population regulation, but our understanding of their function(s) in planktonic systems is severely limited and fragmentary. This project addresses significant knowledge gaps and explores the potential complexities of marine planktonic EV production. This project provides support and training to a female graduate and undergraduate marine sciences students, who are receiving unique opportunities to master new experimental approaches and state-of-the-art research tools that are extremely rare in marine sciences programs. The project supports high school students in marine sciences studies as a part of the summer science camp (www.sigmacamp.org). A female postdoc is also being trained on the project.Using the cosmopolitan and geochemically-important microalga E. huxleyi as a model system, this project tests three major hypotheses to enhance our understanding of the purpose(s) of microalgal EV production. (1) Microalgae produce distinctive types of EVs (ectosomes or exosomes) in response to different environmental conditions, and EV types have definitive functions (stress response, viral defense, intercellular communication, waste disposal). (2) EVs’ cargo is diverse, so their production and release reflect a complex intercellular communication mechanism. (3) Exosome genesis is a multistage process, and its stages are separated in time. Therefore, algal cells may contain a pool of pre-formed EVs loaded with different cargo that are stored internally, and when induced by a sudden change in external conditions are released through the outer membrane. To adequately test these hypotheses requires using single particle analytical methods in addition to ensemble measurements. The investigators are using an assortment of recently developed methods and original experimental approaches developed by our group to investigate EV compositional variability under selected stress conditions. They use single particle Raman microspectroscopy, pulse-chase Stable Isotope Probing, and LC-MSMS for compositional analysis of EVs, and Cryo-EM and AFM for morphological analyses. If experimental data confirm our suspicions, then phytoplankton EVs represent a novel and essentially overlooked mechanism of extracellular interactions that potentially govern a wide range of globally-important processes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

EAGER项目是一项概念验证研究，研究海洋微藻在应对各种环境和生物压力时产生的细胞外囊泡（EV）的组成，起源和动力学。EV是微观脂质包裹的颗粒，几乎从所有细胞类型中自然释放，是各种货物的载体，包括遗传物质（RNA，DNA），蛋白质和脂质。EV已经被各种各样地假设为防御病毒攻击、废物处理机制、应激反应或细胞间通信的手段。海洋微藻是全球碳循环的关键参与者。通过更好地了解控制其种群动态和对环境变化的反应的过程，我们可以开发更好的预测模型来应对全球气候变化。随着气候变化变得越来越明显，了解这些机制的必要性变得越来越紧迫。最近的研究结果表明，EV在海洋浮游植物种群调节中起着关键作用，但我们对它们在浮游系统中的功能的理解是非常有限和零碎的。该项目解决了重大的知识差距，并探讨了海洋生物电动汽车生产的潜在复杂性。该项目为女性研究生和本科海洋科学学生提供支持和培训，他们正在获得独特的机会来掌握新的实验方法和最先进的研究工具，这在海洋科学课程中是非常罕见的。该项目支持高中学生进行海洋科学研究，作为暑期科学夏令营的一部分（www.sigmacamp.org）。一名女博士后也正在接受该项目的培训。huxleyi作为一个模型系统，这个项目测试三个主要的假设，以提高我们的理解的目的（S）微藻EV生产。(1)微藻响应于不同的环境条件产生不同类型的EV（外泌体或外来体），并且EV类型具有确定的功能（应激反应、病毒防御、细胞间通信、废物处理）。(2)电动汽车的货物是多种多样的，因此它们的生产和释放反映了复杂的细胞间通讯机制。(3)外泌体发生是一个多阶段的过程，其阶段在时间上是分开的。因此，藻类细胞可能含有一池预先形成的EV，这些EV装载有储存在内部的不同货物，并且当外部条件突然变化引起时，通过外膜释放。为了充分检验这些假设，除了集合测量外，还需要使用单粒子分析方法。研究人员正在使用各种最近开发的方法和我们小组开发的原始实验方法来研究在选定的应力条件下EV成分的变化。他们使用单粒子拉曼显微光谱、脉冲追踪稳定同位素探测和LC-MSMS进行EV的成分分析，并使用Cryo-EM和AFM进行形态分析。如果实验数据证实了我们的怀疑，那么浮游植物EV代表了一种新的，基本上被忽视的细胞外相互作用机制，可能会管理广泛的全球重要过程。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。