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bioRxiv · 10.1101/2022.08.01.502196

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

Lan, C.·Kim, J.·Ulferts, S.·Aprile-Garcia, F.·Anandamurugan, A.·Grosse, R.·Sawarkar, R.·Reinhardt, A.·Hugel, T.
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Abstract

The formation of biomolecular condensates underpins many cellular processes; however, our current understanding of condensate formation within cells is largely based on observing the final near-equilibrium condensate state. It is less clear how proteins behave before condensates form or at concentrations at which condensation does not occur in cells. Here, we use a combination of fluorescence microscopy and photobleaching analysis to quantify phase separation of negative elongation factor (NELF) in living and stressed cells. We use the recently reported system of stress-induced condensation of NELF in human nuclei as a model to study the behaviour of proteins before condensation. We find that pre-condensate heterogeneous clusters both grow and shrink and are not freely diffusing. Unexpectedly, we also find such small dynamic clusters in unstressed cells in which condensates do not form. We provide a categorisation of small and large clusters based on their dynamics and their response to p38 kinase inhibition. Overall, our data are best explained as non-classical nucleation with a flat free-energy landscape for clusters of a range of sizes and an inhibition of condensation.

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Lan, C., Kim, J., Ulferts, S., Aprile-Garcia, F., Anandamurugan, A., Grosse, R., Sawarkar, R., Reinhardt, A., Hugel, T.. 2022-08-02. Quantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation. https://doi.org/10.1101/2022.08.01.502196

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