Abstract
The mechanisms governing pH regulation in membrane-less organelles (MLOs) are essentially different from those relying on membrane proteins, yet it remains poorly understood due to the difficulty in directly controlling the conditions across the MLOs interface. Here, we develop a coacervate-based, in vitro model to investigate how liquid-liquid phase separation (LLPS) could contribute to pH regulation in MLOs. We construct peptide-based coacervate droplets using microfluidics and find that charged polymers within the coacervates help create uneven H+/OH- distributions, resulting in pH-regionalized microenvironments similar to the nucleolus. More interestingly, such a pH difference weakens or even disappears following the destruction of LLPS, a phenomenon observed in both nucleolus and coacervate droplets. Based on these findings, we demonstrate the ability to finely tune the local pH of the coacervate droplets over a wide range by incorporating enzymes, which can drive and control cascade reactions, and perform basic molecular biology operations such as polymerase chain reaction (PCR) and in vitro transcription and translation reaction (IVTT). This study highlights the role of LLPS in pH modulation within MLOs and provides insights into the potential of coacervates as protocells for broader applications.
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Acknowledgements
This work was supported by the National Key Research and Development Program of China (2024YFA0919100), the National Natural Science Foundation of China (32271383), and the Shenzhen Medical Research Fund (B2401006). We are particularly thankful to the Core Facility of Shanghai Medical College, Fudan University.
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C.W. designed and performed the majority of the experiments, analyzed most of the data, and wrote the manuscript. Z.F. contributed to the analysis of turbidity data. L.Z. carried out part of the cell experiments. L.S. supervised the project, contributed to the experimental design, and co-wrote the manuscript. Y.Z. contributed to the experimental design and manuscript writing.
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Wang, C., Fang, Z., Zhang, L. et al. Coacervate droplets as pH-regionalized protocells. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68980-6
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DOI: https://doi.org/10.1038/s41467-026-68980-6
