Distinct subpopulations of extracellular vesicles are released from the cilia of C. elegans sensory neurons
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Extracellular vesicles (EVs) are shed from primary cilia in multiple systems, yet their role in regulating physiological processes is unclear. As the functionality of an EV is dependent upon its molecular cargo; understanding how cargo is sorted into ciliary EVs is fundamental to understanding their physiological potential. ☐ I discovered that the CLHM-1 ion channel is novel cargo in EVs shed from the cilia of C. elegans sensory neurons. Using TIRF super-resolution microscopy, I showed that tdTomato-tagged CLHM-1 is enriched into a subpopulation of EVs distinct from those containing GFP-tagged PKD-2, a well-characterized EV cargo. Analysis of fluorescent cilia images shows that PKD-2 and CLHM-1 differentially localize to distinct subregions of the cilia, leading to their EV enrichment. ☐ To understand how the cilia achieves discrete ciliary sublocalization and EV enrichment, I examined EV release in kinesin-II loss-of-function mutants and found that individual loss of the kinesin OSM-3 or heterotrimeric kinesin-II had differential effects on release of CLHM-1 and PKD-2 EVs, as well as their ciliary colocalization. Loss of both OSM-3 and heterotrimeric kinesin-II prevented cilia entry and EV inclusion of PKD-2, but not CLHM-1. Deleting the transition zone ciliary diffusion barrier also impacted release of PKD-2, but not CLHM-1, EVs. ☐ To characterize other molecular cargo in the CLHM-1 EV subpopulation, I engineered a chimeric CLHM-1 protein containing a Strep-tag in an extracellular loop domain for affinity isolation. I confirmed that Strep-tagged CLHM-1 localizes to the cilia, is included in EVs, and binds to Streptactin affinity resin, indicating the potential of Strep-tagged CLHM-1 as a tool for isolating intact ciliary EVs. ☐ Ultimately, my work develops C. elegans as an in vivo model for studying protein packaging into EVs shed from cilia and describes TIRF imaging as a novel technique for super-resolution imaging of fluorescently labeled EVs. I discovered that enrichment of protein cargo into ciliary EVs is a consequence of protein ciliary sublocalization. Future works include using super-resolution microscopy to explore other molecular mechanisms that alter protein ciliary distribution and EV enrichment, and to isolate intact EVs to characterize their molecular cargo and physiological potential.
Description
Keywords
C. elegans, Extracellular vesciles, Sensory neurons