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

这个迫切的项目是关于海洋微藻在应对各种环境和生物应激源时产生的胞外小泡(EV)的组成、来源和动力学的概念验证研究。EV是被脂质包裹的微小颗粒，几乎从所有类型的细胞中自然释放，是运输各种货物的工具，包括遗传物质(RNA、DNA)、蛋白质和脂类。电动汽车一直被认为是抵御病毒攻击的一种防御手段，一种废物处理机制，一种压力反应，或一种细胞间沟通的手段。海洋微藻是全球碳循环中的关键角色。通过更好地了解支配其人口动态和对环境变化反应的过程，我们可以开发出更好的对全球气候变化反应的预测模型。随着气候变化变得更加明显，理解这些机制的必要性变得越来越迫切。最近的发现表明，EVS在海洋浮游植物种群调节中发挥着关键作用，但我们对它们在浮游系统中的功能(S)的了解严重有限和支离破碎。该项目解决了重大的知识差距，并探索了海洋浮游式电动汽车生产的潜在复杂性。该项目为海洋科学专业的女研究生和本科生提供支持和培训，她们获得了掌握新的实验方法和最先进的研究工具的独特机会，这些方法在海洋科学课程中极其罕见。作为夏季科学夏令营的一部分，该项目支持高中生学习海洋科学(www.sigmacamp.org)。一名女性博士后也在接受该项目的培训。该项目以具有世界性和地球化学重要性的微藻E.huxleyi作为模型系统，检验三个主要假设，以增强我们对微藻EV生产目的(S)的理解。(1)微藻根据不同的环境条件产生不同类型的EV(胞外体或外体)，EV类型具有明确的功能(应激反应、病毒防御、细胞间通讯、废物处理)。(2)电动汽车的货物种类繁多，其产生和释放反映了复杂的细胞间通讯机制。(3)外切体的形成是一个多阶段的过程，各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电动汽车，这些电动汽车装载着不同的货物储存在内部，当外部条件突然变化时，这些电动汽车通过外膜释放。为了充分检验这些假设，除了整体测量外，还需要使用单粒子分析方法。研究人员正在使用一系列最近开发的方法和我们团队开发的原始实验方法来研究选定应力条件下EV成分的可变性。他们使用单粒子拉曼光谱、脉冲追逐稳定同位素探测和LC-MSMS来分析电动汽车的成分，并使用Cryo-EM和AFM来进行形态分析。如果实验数据证实了我们的怀疑，那么浮游植物电动汽车代表了一种新的、基本上被忽视的细胞外相互作用机制，可能管理着广泛的全球重要过程。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.