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

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 ( <a:math xmlns:a="http://www.w3.org/1998/Math/MathML"> <a:mrow> <a:mi>SOD</a:mi> <a:msub> <a:mn>1</a:mn> <a:mi>bar</a:mi> </a:msub> </a:mrow> </a:math> ) inside stress granules (SGs), comparing the measurements simultaneously to the surrounding cytoplasmic fluid of a single living cell. Surprisingly, <b:math xmlns:b="http://www.w3.org/1998/Math/MathML"> <b:mrow> <b:mi>SOD</b:mi> <b:msub> <b:mn>1</b:mn> <b:mi>bar</b:mi> </b:msub> </b:mrow> </b:math> is not destabilized in SGs relative to the cytoplasm, contrary to expectations from previous 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.