Isolation and Proteomics of Extracellular Microvesicles Shed by Tetrahymena thermophila during Mating

T. thermophila is a ciliated protozoan found in fresh water. Early during sexual reproduction (Fig. 1) they undergo cell adhesion followed by membrane fusion resulting in 100s of pores in the future mating junction. Pores start as small 80 nm protrusions and later expand to accommodate nuclear exchange. During pore expansion we observed microvesicles (EMVs) in the extracellular space of the mating junction (Fig. 2). EMVs were isolated by disrupting pairs of T. thermophila 2‐3 hours into conjugation and performing differential high‐speed centrifugation. The presence of vesicles was confirmed by electron microscopy with negative staining. Proteomics of the isolated vesicles was performed using capillary liquid‐chromatography and tandem mass spectroscopy. The results of proteomic analysis (specifically the presence of proteins associated with ubiquitination and formation of multivesicular bodies), were consistent with a hypothesis suggesting that EMVs represent a “membrane excavation” mechanism that supports the process of pore‐expansion associated with membrane remodeling. We also found evidence of proteasomes, typically associated with cytoplasmic protein recycling. Finally, we found proteins associated with RNAi, suggesting that these vesicles may be involved in down‐regulating small “scan” RNAs that are massively expressed from the germ‐line nucleus during meiosis. All these findings lead us to conclude that EMVs are very likely to represent a complex recycling mechanism targeting cytoplasmic proteins, miRNAs and membrane components during the early stages of conjugation. We are currently pursuing RNA‐SEQ analysis to explore whether or not miRNAs are indeed being expelled from the cell during mating