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

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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.