Thursday, April 3, 2025
4/3/2025
Determining the mechanisms of extracellular vesicle release, function, and clearance - Project Summary/Abstract Cells release extracellular vesicles (EVs) that carry signals to alter cell fate or metabolism, promote invasive behavior, or modulate the immune response. EVs show great potential as diagnostic biomarkers for disease progression, especially in inflammation and cancer. EVs also show promise as a platform for targeted drug delivery, given their ability to signal to and be taken up by cells. Furthermore, EVs can be degraded via phagocytosis, providing metabolites to the engulfing cell. Despite the interest in EVs, basic cell biological knowledge about EV biogenesis, targeting, cargo transfer, and in vivo function is lacking, largely due to the challenges of imaging <250 nm vesicles that lack specific markers. We pioneered a novel labeling technique using degron protection assays to specifically label EVs, which allows us to image EVs in vivo using time-lapse microscopy. We take advantage of the optical transparency and simple genetics of the worm model system C. elegans for gene discovery and have established the first molecular pathway describing how EVs bud from the plasma membrane like viruses. We identified conserved proteins that inhibit EV release in worms and human cells and have generated genetic tools and quantitative assays that enable us to screen for proteins that promote EV release. We also used degron labeling to determine how large EVs and cell corpses are cleared by phagocytosis, revealing novel insights into cargo membrane breakdown and phagolysosomal vesiculation for degradation. Our goals are to determine the molecular interactions of the proteins we identified that regulate lipid asymmetry and EV budding, to determine the role of phosphatidylethanolamine lipids in EV budding, to perform genetic screens using sensitized strains to discover novel proteins involved in EV biogenesis, and thereby define the molecular regulation of EV release. We also plan to use our specific labeling technique to track individual EVs to determine how EVs interact with cells and transfer cargo, providing dynamic insights into their functions from developmental signaling to membrane remodeling. We will also use larger EVs and cell corpses to study how membrane-wrapped cargos are processed inside phagosomes, especially the role of autophagy-associated Atg8/LC3 lipidation in cargo membrane breakdown. Breakdown of the EV membrane in endolysosomes may contribute to EV cargo transfer after uptake. This work has the potential to transform EV production and targeting for drug delivery, as well as to identify key players in EV biology that can be targeted to influence viral and metastatic spread, diverse diseases, and homeostasis. Furthermore, defining the mechanisms of phagocytic breakdown is likely to provide insights into immune modulation and inflammation. Finally, this work on lipid asymmetry is likely to reveal novel aspects of lipid regulation during key processes from cell fusion to cell division. Thus, our vision is to discover how EV release is regulated and use this to determine the functional roles of EVs in vivo while also providing mechanistic insights into diverse proteins and lipids that regulate membrane dynamics.
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