Identifying factors that impact EV biogenesis in C. elegans ciliated sensory neurons
Date
2025
Authors
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Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Small secreted extracellular vesicles (EVs) mediate the intercellular transport of bioactive macromolecules during physiological processes and propagation of pathological conditions. The primary cilium, a sensory organelle protruding from most non-dividing cells, transmits signals by shedding EVs called ectosomes. In this work, I utilize the model organism Caenorhabditis elegans to study different factors regulating ectosome biogenesis and release, with a focus on membrane composition and membrane trafficking. ☐ I found that the C. elegans type I phosphatidylinositol 4-phosphate 5-kinase PPK-1 localizes to and regulates PI(4,5)P2 abundance in the ciliary base, while the C. elegans phosphoinositide 5-phosphatase INPP-1 localizes to and impacts PI(4,5)P2 in the cilium proper of male-specific ray type B (RnB) EV-releasing neurons (EVNs). Using a genetic approach to manipulate PI(4,5)P2 abundance in ciliary compartments of worms expressing fluorescently labeled EV cargos, we discovered that high PI(4,5)P2 increases budding of EVs derived from the cilium tip, but inhibits biogenesis of an EV subpopulation shed from the PCMC. This altered EV biogenesis is not accompanied by a change in cilium length, suggesting that increasing PI(4,5)P2 in the cilium proper can serve as a mechanism to enhance EV ectocytosis from the distal tip without inducing ciliary decapitation. ☐ I have also discovered a novel role for the Rab GTPase, RAB-28, in ciliated sensory neurons. I found that disruption of rab-28, but not the BBSome complex that traffics RAB-28 into the cilium proper, decreases the release of CLHM-1-containing EVs into the environment due to an increase in phagocytosis of these vesicles by the glial cells ensheathing the sensory neurons. This contrasts with the biogenesis of PKD2-containing EVs, which is not impacted by the loss of rab-28 or bbs-8. This is likely because PKD-2 EVs are not subject to the same glial phagocytosis mechanism as they bud off of the environmentally exposed distal tip of cilia. ☐ Lastly, I showcase an advanced sample preparation technology for proteomic analysis of C. elegans, which is notoriously difficult to process, using methanol fixation. We developed an efficient, effective, and economical (E3) technology for global proteome analysis using on-filter in-cell (OFIC) processing. Compared with other existing methods, it bypasses the need for cell lysis by directly digesting proteins in methanol-fixed cells. This significantly reduces the number of steps and amount of time required for sample preparation and streamlines all proteomic processing in a single device. As a proof of concept, I then successfully utilized this method to compare the proteomes of wild-type and mutant C. elegans, focusing on how the proteome is impacted by loss of evolutionarily conserved Cu/Zn superoxide dismutase 1 (SOD1), a critical player in cellular defense via catalysis of superoxide radicals into less harmful oxygen and hydrogen peroxide. OFIC processing is the simplest workflow for C. elegans proteomics and can be used to answer biologically significant questions, including how altered EV shedding impacts the proteome of cells that release and uptake EVs.
Description
Keywords
C. elegans, Cilia, Extracellular vesicles, Phospholipids, Proteomics