Quantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation

Chenyang Lan, Juhyeong Kim, Svenja Ulferts, Fernando Aprile-Garcia, Sophie Weyrauch, Abhinaya Anandamurugan, Robert Grosse, Ritwick Sawarkar, Aleks Reinhardt () and Thorsten Hugel ()
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Chenyang Lan: University of Freiburg
Juhyeong Kim: University of Freiburg
Svenja Ulferts: University of Freiburg
Fernando Aprile-Garcia: Max Planck Institute of Immunobiology and Epigenetics
Sophie Weyrauch: University of Freiburg
Abhinaya Anandamurugan: University of Freiburg
Robert Grosse: University of Freiburg
Ritwick Sawarkar: University of Cambridge
Aleks Reinhardt: University of Cambridge
Thorsten Hugel: University of Freiburg

Nature Communications, 2023, vol. 14, issue 1, 1-8

Abstract: Abstract Our current understanding of biomolecular condensate formation is largely based on observing the final near-equilibrium condensate state. Despite expectations from classical nucleation theory, pre-critical protein clusters were recently shown to form under subsaturation conditions in vitro; if similar long-lived clusters comprising more than a few molecules are also present in cells, our understanding of the physical basis of biological phase separation may fundamentally change. Here, we combine fluorescence microscopy with photobleaching analysis to quantify the formation of clusters of NELF proteins in living, stressed cells. We categorise small and large clusters based on their dynamics and their response to p38 kinase inhibition. We find a broad distribution of pre-condensate cluster sizes and show that NELF protein cluster formation can be explained as non-classical nucleation with a surprisingly flat free-energy landscape for a wide range of sizes and an inhibition of condensation in unstressed cells.

Date: 2023
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DOI: 10.1038/s41467-023-40540-2

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