Abstract

The nucleolus is a membraneless organelle embedded in chromatin solution inside the cell nucleus. It has recently been reported that nucleoli in frog oocytes and C. elegans embryos form by liquid-liquid phase separation [1,2]. While fusion of nucleoli in human cells has been observed, it is unclear if they are liquid-like or solid-like in nature [3,4]. Moreover, the role of the chromatin polymer in the facilitation of nucleolar fusion is completely unknown. In this work, we investigate the kinetics of the nucleolar fusion in order to determine its physical nature, i.e. aggregation of solid-like particles vs. coalescence of liquid-like droplets. We monitor and analyze the nucleolar shape during fusion events and find it consistent with a coalescence of liquid droplets. Further, we analyzed nucleolar fusion events and found that after fusion, the eccentricity of the new nucleolus decreases, indicating the nucleolus undergoes a structural change and behaves as a liquid-like structure. Historically, the nucleolar assembly in mammalian cells was believed to complete within two hours after mitosis, when the final number of nucleoli forms via de novo assembly as well as fusion events [3]. However, we found that fusion events can also occur later in the cell cycle. To explore the interaction of nucleoli with chromatin, we altered chromatin state using biochemical perturbations. We found that chromatin is indeed involved in the positioning, shape and alignment of nucleoli inside the cell nucleus. 1. Brangwynne, CP et al., Proc. Natl. Acad. Sci., 4434, 2011 2. Berry, J, et. al. Proc. Natl. Acad. Sci., E5237, 2015 3. Savino, TM, et al. J. Cell Biol.,1097, 2001 4. Farley, KI, et al. Chromosoma, 323, 2015

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