The role of anterograde IFT motors and lipid scramblases in the organization of extracellular vesicle carbo into ciliary microdomains

Abstract

Extracellular vesicles (EVs) are lipid enclosed containers of signaling molecules, metabolic products, various proteins and RNAs that play a wide variety of roles in cellular processes. Although they are likely released from most cell types, the mechanisms underlying extracellular vesicle (EV) cargo sorting and biogenesis have not yet been fully elucidated due in part to the reliance in the field on in vitro models. Utilizing a C. elegans ciliated sensory neuron EV model that releases two separate subpopulations of EVs containing either CLHM-1::tdTomato or PKD-2::GFP, we were able to shed light on a possible sorting mechanism via physical separation of EV cargo into domains within the cilia. Intraflagellar transport (IFT) is required for construction of the middle and distal ciliary segments, proper protein localization, and EV release. Anterograde IFT motors OSM-3 and KLP-11 are required for the normal release of PKD-2 containing EVs, but not CLHM-1 containing EVs. Anterograde IFT is also required for proper localization of PKD-2 in the distal segment of the cilia, and abundance of CLHM-1 within the periciliary membrane compartment. The transition zone, which separates the cilia proper from the periciliary membrane compartment, is also required for normal EV subpopulation abundance. Transition zone breakdown caused by mks-5 and subsequent disorganization of proteins within the cilia correlates with an increase in colocalized EVs containing both CLHM-1::tdTomato and PKD-2::GFP. ☐ The C. elegans EV model can be used to study the impact of Ca2+ on EV biogenesis. Addition of the calcium ionophore ionomycin corresponded with an increased release in CLHM-1 containing EVs. This calcium-induced release of CLHM-1 containing EVs may occur via the scramblase ANOH-1. Scramblases are hypothesized to play a role in EV biogenesis by inducing membrane curvature in response to a chemical signal, such as calcium influx. In The Role of Anterograde IFT Motors and Lipid Scramblases in the Organization of Extracellular Vesicle Carbo into Ciliary Microdomains, anoh-1 mutants release significantly fewer CLHM-1 containing EVs when compared to WT. Preliminary data indicates that biogenesis of the PKD-2::GFP containing subpopulation does not required ANOH-1. Further investigation is needed into the role of calcium in EV biogenesis.