The Characterization and Tuning of Nucleolar Compartmental Dynamics using the Cryptochrome 2 (CRY2-olig) Optogenetic System

Abstract

Ribonucleoprotein (RNP) bodies are membrane-less intracellular organelles that have been shown to behave as liquid droplets and assemble via liquid-liquid phase separation. The nucleolus, a type of RNP body located in the nucleus, is responsible for the transcription of ribosomal DNA, processing, and assembly of ribosomal RNA. Electron microscopy shows that the nucleolus is partitioned into three different compartments, granular compartment (GC), dense fibrillar compartment (DFC), and fibrillar compartment (FC). These compartments are arranged in a layered fashion with the inner-most compartment being the FC and the outer-most compartment being the GC. The compartmental liquid-like dynamics differ between compartments possibly due to differing enzymatic responsibilities. Additionally, it has been shown that Xenopus laevis oocytes have an actin meshwork to stabilize the internal structure of nucleoli and prevent largescale fusion under physiological conditions. Although the nucleolus has been researched extensively in the past, the way in which biophysical properties are affected by molecular interactions of the different nucleolar compartments is still unclear. Here, I use optogenetic manipulation of a reversible, blue light induced oligomerizing protein, Cryptochrome 2 (CRY2-olig), to spatio-temporally control molecular interactions between nucleolar proteins in the large X. laevis oocyte nucleus. I also use fluorescence recovery after photobleaching (FRAP), actin disruption techniques, and fluorescent dextran injection in tandem with CRY2-olig to characterize the impact of light activated CRY2-olig oligomerization on the mobility of proteins in nucleolar compartments. We found that CRY2-olig activation impacts nucleoli in the following ways: 1) slows intra-compartmental nucleolar liquid-like dynamics, 2) affects inter-compartmental dynamics when an adjacent compartment is activated, 3) alters the nucleolar meshwork, 4) hinders nucleolar fusion, and 5) impacts nucleolar rRNA output. Ultimately, I show that the molecular and bulk properties of X. laevis nucleoli can be controlled by 488 nm light activation. These findings will shed light on the physiology of the nucleolus as a multi-phase liquid organelle dependent upon compartmental interactions and an appropriately sized nucleolar meshwork for efficient ribosomal biogenesis and subsequent transfer of genetic information.