Researcher(s)
- Krisha Parekh, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Jessica Tanis, Biological Sciences, University of Delaware
Abstract
Extracellular vesicles (EVs) assist in the transport of biological macromolecules between nearly all cell types. The process of EV release is still relatively unknown. It is important to define underlying factors involved in the sorting of EV cargoes and release of EV subpopulations in order to better understand their impacts on pathophysiological processes underlying neurodegenerative diseases and tumor development. In Caenorhabditis elegans, EVs are shed from neuron primary cilia, microtubule-based organelles that play a role in signal transduction and transmission. Two specific EV subpopulations are released: one that contains the TRP polycystin channel PKD-2 and another that contains the ion channel CLHM-1. To visualize the EVs that have been shed, CLHM and PKD-2 have been tagged with green fluorescent protein (GFP). These EV subpopulations are differentially shed, as PKD-2 EVs are released from the ciliary distal tip, while CLHM-1 EVs are released from the ciliary base. Phosphoinositides bind proteins to regulate ciliary localization and endocytic and exocytic processes. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) is a phospholipid selectively found in the ciliary base. In prior work, we established that the conserved phosphatase, inositol phosphate-1-phosphatase (INPP-1), impacts our release of PKD-2::GFP EVs, defining a new role for PI(4,5)P2 in EV release. From here, we aimed to describe the impact of the phosphatase CIL-1, another protein that breaks down PIP2, on EV biogenesis. We found that CIL-1::GFP preferentially localized to the ciliary base. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we observed that cil-1(my15) mutant animals had no impact on the release of PKD-2::GFP and CLHM-1::GFP-containing EVs. This work provides insight into the complex mechanisms behind EV biogenesis and release and helps elucidate the role of PIP2 in EV shedding.