Intracellular Phase Transitions: The Role of Fibrillarin in Nucleolar Assembly

Abstract

Nucleoli are non-membrane bound organelles formed within the nucleus of eukaryotes that serve as the site of rRNA transcription, processing, and ribosome subunit assembly. Interestingly, these intracellular bodies self-assemble from high concentrations of proteins in the nucleoplasm: this process is hypothesized to occur due to a liquid-liquid phase transition. The snoRNP protein fibrillarin is often used as a marker of nucleoli and is thought to be involved in the nucleolar assembly process. This study will investigate the role of fibrillarin in the phase transitions hypothesized to govern nucleolar assembly. The process of nucleolar assembly includes the formation of many fluorescent protein droplets and their combining to form two nucleoli in each nucleus. First, nucleolar assembly in the wild type will be characterized by size, number, and intensity of nucleoli. Then, fibrillarin protein will be knocked down using RNAi, RNA interference, and assembly will again be characterized. The nucleoli after a knockdown of fibrillarin will be imaged using two different markers of nucleoli, FIB-1::GFP and DAO-5::GFP. The data from this study indicates that there exists no phase boundary with decreasing fibrillarin concentration, meaning that fibrillarin is not essential for the formation of fluorescent protein droplets or nucleoli. It was shown, however, that a decrease in fibrillarin caused smaller size of nucleoli and fewer fluorescent droplets formed. These results indicate that while fibrillarin may not be essential for nucleolar formation, it does have an effect on the size and number of nucleoli and fluorescent protein droplets.