Phosphatidylinositol (4,5)-bisphosphate Impacts Ectosome Shedding from Ciliated Sensory Neurons.

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. Although the ciliary membrane is continuous with the plasma membrane, it exhibits unique phospholipid distribution, with levels of phosphatidylinositol 4,5-bisphosphate PI(4,5)P high in the periciliary membrane compartment (PCMC), but low in the cilium proper and distal tip. The functional impact of PI(4,5)P on ectosome biogenesis is not known. In sensory neurons, different populations of ectosomes are shed from the PCMC and cilium distal tip. We used a genetic approach to increase PI(4,5)P in the PCMC by overexpressing the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K1) PPK-1 or in the cilium proper through deletion of the phosphoinositide 5-phosphatase (INPP5E) , then imaged released EVs that carried different fluorescently-tagged cargos. We discovered that high PI(4,5)P differentially affected shedding of distinct ectosome populations from ciliary subcompartments, decreasing biogenesis of EVs from the PCMC, but increasing budding from the cilium distal tip. While manipulating PI(4,5)P also impacted the trafficking, localization, and abundance of EV cargos in the cilium, localization of these proteins to distinct subsets of ectosome was unchanged, suggesting that PI(4,5)P does not impact cargo sorting. Further, the PI(4,5)P-dependent increase in ectosome shedding from the distal tip did not alter cilium length. Thus, altering PI(4,5)P serves as a mechanism to specifically regulate biogenesis of ectosomes shed in response to physiological stimulus.