Linking Nucleolar Biophysics to Function

Abstract

The cell nucleus contains a large number of non-membrane bound bodies that play important roles in the spatio-temporal regulation of gene expression. The nucleolus is a nuclear body that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). While studies have shown that nucleoli have a viscoelastic material nature and likely assembles through liquid-liquid phase separation, how its material properties relate to its functionality are not well known. In addition, while nucleolar proteins have been shown to phase separate in-vitro, understanding of how this phase separation works in the multi-component nucleolus in the cell is also not well understood.

In this thesis, we describe first how we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres, but nonetheless permit continued internal molecular mobility of proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors, and a depletion of smaller rRNA precursors. Secondly, we also describe how proteins partition as a result of multi-component phase separation into membrane-less condensates. We focus specifically on how nucleolar protein partitioning in the granular compartment of the nucleolus is related to rRNA processing into ribosomes. Through measurements of partition coefficients in the living cell, we address the interplay between nucleolar protein interactions, phase separation, and the processing of rRNA complexes. We employ a rich diversity of tools and organisms in order to elucidate some biophysical characteristics of the nucleolus (in terms of material properties and phase separation) and its relationship to functionality.