Poster abstracts

Poster number 43 submitted by Mary English

Development of imaging tools to map the pH and redox environment of ribonucleoprotein biomolecular condensates

Mary English (Chemistry, University of Pittsburgh), Wade Schnorr (Chemistry, University of Pittsburgh), Michael Dorogan (Chemistry, University of Pittsburgh), Alex Deiters (Chemistry, University of Pittsburgh), Seth Childers (Chemistry, University of Pittsburgh)

Abstract:
Biomolecular condensates organize biochemical reactions by concentrating proteins and nucleic acids within phase separated compartments. While their composition and material properties have been extensively studied, less is known about their pH and redox state, which could influence condensate functions. We developed imaging approaches to spatially resolve the pH and redox environments of ribonucleoprotein (RNP) biomolecular condensates formed by bacterial RNA degradosome-associated protein RNase E (RNE). To measure condensate pH , we developed a HaloTag-based fluorescent probe and compared it to three approaches. These methods found that the RNase E condensate assembled in pH 7.5 buffer using a conjugated Halo tag exhibited a near neutral effective pH (7.4), while in contrast free dye (6.7), pHluorin (6.5) were more acidic and bioconjugated dye (10.8) was more alkaline. We interrogate and discuss the impacts and caveats of the local environment adjacent to fluorescent dye may impact pH readings. We next considered the redox conditions of this environment, given associated BR-body clients may influence redox state (e.g. GSH reductase, superoxide dismutase, and thioredoxin reductase). Using a genetically encoded redox-sensitive GFP (roGFP) reporter fused to the CTD of RNE, we found that the condensate interior was more reduced relative to the cytoplasm.With moderate concentrations of H2O2 and CuSO4, the BR-bodies maintained their reducing environment, despite more oxidizing cytoplasmic conditions which may be critical for maintaining degradosome activity in redox stress. In summary, these findings demonstrate that RNP condensates are chemically distinct pH and redox microenvironments, that may be critical for activating, protecting or arresting client protein functions. Overall, these imaging approaches provide a framework for investigating how physicochemical properties of diverse RNP condensates influence RNA-processing and stress response.

References:
Schnorr, W.E., Sasazawa, M., Dzurik, K.G. et al. Bacterial ribonucleoprotein bodies maintain an acidic pH environment as a mechanism of enzyme regulation. Nat Commun (2026). https://doi.org/10.1038/s41467-026-76859-9
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers
of cellular biochemistry. Nat Rev Mol Cell Biol 18, 285-298 (2017).
https://doi.org:10.1038/nrm.2017.7
Muthunayake, N. S., Tomares, D. T., Childers, W. S. & Schrader, J. M. Phase-separated
bacterial ribonucleoprotein bodies organize mRNA decay. Wiley Interdiscip Rev RNA 11,
e1599 (2020). https://doi.org:10.1002/wrna.1599

Keywords: condensates, redox, pH