Presenting author: Eric S. Cole1
Tetrahymena thermophila undergo mating that involves adhesion of two cell partners, formation of a mating junction between conjugating cells, and the reciprocal exchange of nuclei across the mating junction. Cell adhesion happens early during pair formation and is followed by cell fusion initiated at hundreds of sites in the future exchange junction. Fusion loci begin as small membrane protrusions that span the intercellular gap, make contact and fuse with membrane of the mating partner. The resulting junction pores expand to create a membrane "curtain" that accommodates bi-directional nuclear exchange. During pore expansion one observes microvesicles extruded into the extracellular space within the mating junction. This study aims to understand the role of these extracellular microvesicles (EMVs). Two hypotheses were explored: 1) EMVs could participate in membrane excavation during pore expansion; 2) EMVs could be involved in signaling between mating cells relevant to advancement of the developmental program. EMVs were isolated by disrupting mating pairs of T. thermophila 2-3 hours into conjugation and performing differential high-speed centrifugation. The presence of vesicles was confirmed by negative staining and transmission electron microscopy. Both RNA and protein were detected in purified EMV material. Protein identification from isolated vesicles was performed using capillary liquid-chromatography and tandem mass spectrometry. The results of proteomic analysis (specifically the presence of proteins associated with ubiquitination and formation of multivesicular bodies), were consistent with the hypothesis suggesting that EMVs participate in membrane remodeling during pore-expansion. Proteasome components, typically associated with protein recycling, were also found. Finally, we found Argonaute proteins normally associated with RNAi, suggesting that EMVs could be involved in down-regulating the small "scan" RNAs expressed from the germ-line nucleus early during meiosis. These findings suggest that EMVs represent a complex recycling mechanism targeting cytoplasmic proteins, miRNAs and membrane components during early stages of conjugation. Preliminary evidence also suggests that EMVs secreted early during early conjugation may play a role in cell signaling with developmental consequences. We are currently pursuing RNA analysis of the EMV contents, and following up on their potential role as mediators of developmental signaling. .