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Protein Folding Stability Simultaneously Imaged in Stress Granules and the Cytoplasm of a Single Living Cell

Mailin Becker1,2,3,*, Nirnay Samanta3,*, Erik Tsvetaev3, Miká Vollet3, Sara S. Ribeiro1,2,3, and Simon Ebbinghaus1,2,3,†

  • 1Chair of Biophysical Chemistry, Ruhr University Bochum, Bochum, Germany
  • 2Research Center Chemical Sciences and Sustainability, Research Alliance Ruhr Bochum, Germany
  • 3Institute of Physical and Theoretical Chemistry, TU Braunschweig, Braunschweig, Germany

  • *These authors contributed equally to this work.
  • †Contact author: simon.ebbinghaus@rub.de
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PRX Life 4, 033005 – Published 16 July, 2026

DOI: https://doi.org/10.1103/dsw6-t4qf

Abstract

Protein folding is governed by distinct subcellular environments with unique biochemical compositions. Unfavorable conditions can lead to unfolding and aggregation, and cells deploy spatially targeted protein quality control mechanisms to promote folding. Understanding how proteostasis is balanced across diverse cellular conditions requires novel techniques that measure protein folding with high spatiotemporal resolution in living cells. Here we introduce confocal fast relaxation imaging (cFReI), which eliminates wide-field signal averaging compared to previous fast relaxation imaging approaches and enables quantification of biomolecular folding stability with enhanced axial resolution in three dimensions. We apply cFReI to monitor the folding stability of a destabilized monomeric apo variant of SOD1 (SOD1bar) inside stress granules (SGs), comparing the measurements simultaneously to the surrounding cytoplasmic fluid of a single living cell. Surprisingly, SOD1bar is not destabilized in SGs relative to the cytoplasm, contrary to expectations from previous in vitro studies. The study demonstrates that cFReI is a pioneering technique for measuring folding kinetics and thermodynamics of fluorescently labeled biomolecules in different biomolecular condensates compared to their respective intracellular fluids.

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Collection on Physics of Biomolecular Condensates

From the fundamentals of phase separation, dynamics and structure of condensates in biological settings, to advanced imaging methods, this special collection presented by PRX Life showcases exciting and timely work on the physics of biomolecular condensates.

This Invited Collection is coordinated by PRX Life Associate Editor Christoph Weber, in consultation with Jerelle Joseph, Simon Alberti, and Tanja Mittag. Every article published in this collection underwent a rigorous peer review process, adhering to the same high standards applied to all papers handled by the journal. The PRX Life editorial team managed the peer review and made all editorial decisions.

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PRX Life 4, 033005– Published 16 July, 2026
  • Received 9 September 2025
  • Accepted 12 June 2026
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