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Composition dependent phase separation underlies directional flux through the nucleolus

View ORCID ProfileJoshua A. Riback, Lian Zhu, Mylene C. Ferrolino, Michele Tolbert, Diana M. Mitrea, View ORCID ProfileDavid W. Sanders, Ming-Tzo Wei, View ORCID ProfileRichard W. Kriwacki, View ORCID ProfileClifford P. Brangwynne
doi: https://doi.org/10.1101/809210
Joshua A. Riback
1Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA
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Lian Zhu
1Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA
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Mylene C. Ferrolino
2Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38103, USA
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Michele Tolbert
2Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38103, USA
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Diana M. Mitrea
2Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38103, USA
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David W. Sanders
1Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA
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Ming-Tzo Wei
1Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA
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Richard W. Kriwacki
2Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38103, USA
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  • For correspondence: cbrangwy@princeton.edu richard.kriwacki@stjude.org
Clifford P. Brangwynne
1Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA
3Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA
4Howard Hughes Medical Institute
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  • For correspondence: cbrangwy@princeton.edu richard.kriwacki@stjude.org
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Abstract

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3–6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7–9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.

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Posted October 22, 2019.
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Composition dependent phase separation underlies directional flux through the nucleolus
Joshua A. Riback, Lian Zhu, Mylene C. Ferrolino, Michele Tolbert, Diana M. Mitrea, David W. Sanders, Ming-Tzo Wei, Richard W. Kriwacki, Clifford P. Brangwynne
bioRxiv 809210; doi: https://doi.org/10.1101/809210
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Composition dependent phase separation underlies directional flux through the nucleolus
Joshua A. Riback, Lian Zhu, Mylene C. Ferrolino, Michele Tolbert, Diana M. Mitrea, David W. Sanders, Ming-Tzo Wei, Richard W. Kriwacki, Clifford P. Brangwynne
bioRxiv 809210; doi: https://doi.org/10.1101/809210

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