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 has been tagged with tdTomato, and PKD has 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. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) is a phospholipid that regulates various cellular processes like signal transduction and vesicular trafficking and transport. Phosphatidylinositol 4,5 bisphosphate 5-phosphatase CIL-1 is a protein that breaks down PIP2. To better understand the roles of PIP2 in EV biogenesis, I would like to determine if the loss of cil-1 affects the shedding of CLHM-1 EVs, PKD-2 EVs, or both subpopulations. Genetic crosses with C. elegans first had to be conducted to produce nematodes that are homozygous for the cil-1 mutation and the transgenes that express the EV cargoes tagged with fluorescent proteins. Additionally, an endogenous reporter of CIL-1 has been created with a Neon Green fluorescent tag to determine protein localization. These tools will provide insight into the complex mechanisms behind EV biogenesis and release, and help to elucidate the role of PIP2 in EV shedding.