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

Author

Listed:
  • Chenyang Lan

    (University of Freiburg
    University of Freiburg
    PicoQuant GmbH)

  • 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
    University of Freiburg
    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
    University of Freiburg)

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.

Suggested Citation

  • Chenyang Lan & Juhyeong Kim & Svenja Ulferts & Fernando Aprile-Garcia & Sophie Weyrauch & Abhinaya Anandamurugan & Robert Grosse & Ritwick Sawarkar & Aleks Reinhardt & Thorsten Hugel, 2023. "Quantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation," Nature Communications, Nature, vol. 14(1), pages 1-8, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-40540-2
    DOI: 10.1038/s41467-023-40540-2
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    References listed on IDEAS

    as
    1. Elie Dolgin, 2022. "The shape-shifting blobs that shook up cell biology," Nature, Nature, vol. 611(7934), pages 24-27, November.
    2. Lisa Streit & Timo Kuhn & Thomas Vomhof & Verena Bopp & Albert C. Ludolph & Jochen H. Weishaupt & J. Christof M. Gebhardt & Jens Michaelis & Karin M. Danzer, 2022. "Stress induced TDP-43 mobility loss independent of stress granules," Nature Communications, Nature, vol. 13(1), pages 1-18, December.
    3. Victoria Tianjing Yan & Arjun Narayanan & Tina Wiegand & Frank Jülicher & Stephan W. Grill, 2022. "A condensate dynamic instability orchestrates actomyosin cortex activation," Nature, Nature, vol. 609(7927), pages 597-604, September.
    4. Shunsuke F. Shimobayashi & Pierre Ronceray & David W. Sanders & Mikko P. Haataja & Clifford P. Brangwynne, 2021. "Nucleation landscape of biomolecular condensates," Nature, Nature, vol. 599(7885), pages 503-506, November.
    5. W. Michael Babinchak & Benjamin K. Dumm & Sarah Venus & Solomiia Boyko & Andrea A. Putnam & Eckhard Jankowsky & Witold K. Surewicz, 2020. "Small molecules as potent biphasic modulators of protein liquid-liquid phase separation," Nature Communications, Nature, vol. 11(1), pages 1-15, December.
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    Cited by:

    1. Furqan Dar & Samuel R. Cohen & Diana M. Mitrea & Aaron H. Phillips & Gergely Nagy & Wellington C. Leite & Christopher B. Stanley & Jeong-Mo Choi & Richard W. Kriwacki & Rohit V. Pappu, 2024. "Biomolecular condensates form spatially inhomogeneous network fluids," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    2. Mrityunjoy Kar & Laura T. Vogel & Gaurav Chauhan & Suren Felekyan & Hannes Ausserwöger & Timothy J. Welsh & Furqan Dar & Anjana R. Kamath & Tuomas P. J. Knowles & Anthony A. Hyman & Claus A. M. Seidel, 2024. "Solutes unmask differences in clustering versus phase separation of FET proteins," Nature Communications, Nature, vol. 15(1), pages 1-21, December.

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