Adaptive metal ion transport and metalloregulation-driven differentiation in pluripotent synthetic cells

nature chemistry Article https://doi.org/10.1038/s41557-024-01682-y Adaptive metal ion transport and metalloregulation-driven differentiation in pluripotent synthetic cells Received: 19 July 2023 Accepted: 28 October 2024 Published online: 23 December 2024 Check for updates Sayuri L. Higashi 1,2,3,4,6, Yanjun Zheng1,6, Taniya Chakraborty 1,6, Azadeh Alavizargar 5, Andreas Heuer 5 & Seraphine V. Wegner 1 Pluripotent cells can yield different cell types determined by the specific sequence of differentiation signals that they encounter as the cell activates or deactivates functions and retains memory of previous inputs. Here, we achieved pluripotency in synthetic cells by incorporating three dormant apo-metalloenzymes such that they could differentiate towards distinct fates, depending on the sequence of specific metal ion transport with ionophores. In the first differentiation step, we selectively transported one of three extracellular metal ion cofactors into pluripotent giant unilamellar vesicles (GUVs), which resulted in elevation of intracellular pH, hydrogen peroxide production or GUV lysis. Previously added ionophores suppress transport with subsequent ionophores owing to interactions among them in the membrane, as corroborated by atomistic simulations. Consequently, the addition of a second ionophore elicits a dampened response in the multipotent GUV and a third ionophore results in no further response, reminiscent of a terminally differentiated GUV. The pluripotent GUV can differentiate into five final fates, depending on the sequence in which the three ionophores are added. Cells possess an extraordinary capacity to respond and adapt to changes in their surroundings by activating specific pathways and retaining a memory of prior inputs1. Cell differentiation stands out as an exceptional illustration of this adaptability. Initially identical pluripotent cells progressively acquire new functionalities while concurrently suppressing other dormant ones. Throughout the course of differentiation, pluripotent cells progressively commit to a specific fate, influenced by the temporal sequence of various signals in the extracellular environment and the memory encoded in gene networks and posttranslational processes2. As a result, they gradually lose their plasticity, transitioning first into multipotent and ultimately into terminally differentiated cells that no longer respond to previous differentiation cues. Synthetic cells, assembled from molecular components, are already able to mimic various functions associated with living cells3,4, encompassing but not limited to growth5, division5–9, reproduction10–12, differentiation13, metabolism14,15, communication16 and social interactions within communities of synthetic and living cells17–21. However, replicating the adaptive multistage differentiation observed in pluripotent cells remains elusive. Towards this goal, lipid-based synthetic cells with cell-free protein expression have been differentiated in multiple steps through the activation of embedded gene circuits with vesicle fusion13 and along diffusion gradients formed in emulsion-based compartments22. Moreover, non-lipid-based protocells can replicate mechanisms of multicellular differentiation by altering their morphology, membrane permeability and enzymatic 1 Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany. 2Institute for Advanced Study, Gifu University, Gifu, Japan. 3Center for One Medicine Innovative Translational Research, Gifu University, Gifu, Japan. 4United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan. 5Institute for Physical Chemistry, University of Münster, Münster, Germany. 6These authors contributed equally: Sayuri L. Higashi, Yanjun Zheng, Taniya Chakraborty. e-mail: Nature Chemistry | Volume 17 | January 2025 | 54–65 54 Article activity when exposed to unidirectional or counter-directional chemical gradients23. In the context of differentiation in synthetic cells, most examples hinge on cell-free protein synthesis and the differential expression of proteins depending on vesicle fusion13 or diffusion gradients of membrane-permeable small molecules22. Moreover, the morphological differentiation of coacervate-based synthetic protocells has been achieved along artificial morphogen gradients without relying on protein production23. Beyond differentiation in synthetic cells, examples of adaptive behaviour are found only in the context of morphological alterations24,25, positioning in the environment26 and cell-to-cell communication27,28. To achieve a pluripotent synthetic cell that can differentiate towards various fates would require one to orthogonally activate different functions within a single synthetic cell and have these co-regulate each other with a gradual loss of plasticity. During cellular differentiation, signalling events across the membrane assume central importance as cells render themselves insensitive to signals along the way by downregulating the expression of cognate receptors29. Crucially, signal transduction across the membrane necessitates high specificity and preferential amplification30. Therefore, adaptive membrane transport, wherein transport across the membrane changes with the history of inputs, provides a unique opportunity for mimicking the differentiation of cells. In this context, reported lipid-based synthetic cells have relied on artificial gene circuits through the use of programmable DNA sequences, along with semipermeable substrates and/or non-specific transporters such as α-haemolysin, to trigger specific cellular functions13,24,31. Yet, replicating specific and adaptive signalling has not been possible due to the lack of dedicated transmembrane receptors and transporters that could specifically transport molecules and/or differentially activate internal pathways. In this work, we demonstrate the selective and adaptive activation of different enzymatic reactions within pluripotent giant unilamellar vesicle (GUV)-based synthetic cells. In particular, we rely on three different metal ions—Ni2+, Cu2+ and Ca2+—as external signals, which are selectively transported into the GUVs with specific ionophores. Subsequently, these metal ions differentially activate apo-metalloenzymes by using these metal ion cofactors. Depending on the specific enzymatic reaction activated, the synthetic cell displays different behaviours, such as increased intracellular pH, hydrogen peroxide (H2O2) production or cellular lysis. The ionophores serve as decision factors of the cell’s fate, as the first activated enzyme sets the synthetic cell’s fate and suppresses the subsequent activation of the other pathways. The first differentiation step induced by a specific metal ion transport is thereby deterministic, endowing the synthetic cell with specialized capabilities while concurrently losing dormant potential (Fig. 1a). Results and dis (...truncated)