Mukwaya Vincent, Yu Xiaolei, Yang Shuo, Mann Stephen, Dou Hongjing
State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Institute of Composite Materials, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom.
Proc Natl Acad Sci U S A. 2025 Apr;122(13):e2419507122. doi: 10.1073/pnas.2419507122. Epub 2025 Mar 24.
Liquid-liquid phase separation (LLPS) has been achieved in various cytomimetic (protocell) models, but controlling molecular condensation using noninert crowders to systematically alter protocell function remains challenging. Intracellular ATP levels influence protein-protein interactions, and dysregulation of ATP can alter cellular crowding dynamics, thereby disrupting the normal formation or dissolution of condensates. Here, we develop a membranized protocell model capable of endogenous LLPS and liquid-gel-like phase separation through precise manipulation of intermolecular interactions within semipermeable polysaccharide-based microcapsules (polysaccharidosomes, P-somes), prepared using microtemplate-guided assembly. We demonstrate that intraprotocellular diffusion-mediated LLPS can be extended into the liquid-gel-like domain by the uptake of the biologically active crowder ATP, resulting in a range of modalities dependent on the fine-tuning of molecular condensation. Endogenous enzyme activity in these crowded polysaccharidosomes is enhanced compared to free enzymes in solution, though this enhancement diminishes at higher levels of intraprotocellular condensation. Additionally, increased molecular crowding inhibits intraprotocell DNA strand displacement reactions. Our findings introduce an expedient and optimized approach to the batch construction of membranized protocell models with controllable molecular crowding and functional diversity. Our mix-incubate-wash protocol for inducing endogenous LLPS in membranized protocells offers potential applications in microreactor technology, environmental sensing, and the delivery and sustained release of therapeutics.
液-液相分离（LLPS）已在各种细胞模拟（原细胞）模型中实现，但使用非惰性拥挤剂控制分子凝聚以系统地改变原细胞功能仍然具有挑战性。细胞内ATP水平会影响蛋白质-蛋白质相互作用，而ATP的失调会改变细胞拥挤动力学，从而破坏凝聚物的正常形成或溶解。在这里，我们开发了一种膜化原细胞模型，通过对基于半透性多糖的微胶囊（多糖体，P-体）内的分子间相互作用进行精确操纵，能够实现内源性LLPS和液-凝胶样相分离，该微胶囊是使用微模板引导组装制备的。我们证明，通过摄取生物活性拥挤剂ATP，原细胞内扩散介导的LLPS可以扩展到液-凝胶样区域，从而产生一系列取决于分子凝聚微调的模式。与溶液中的游离酶相比，这些拥挤多糖体中的内源性酶活性有所增强，尽管在原细胞内凝聚水平较高时这种增强会减弱。此外，增加的分子拥挤会抑制原细胞内DNA链置换反应。我们的研究结果引入了一种便捷且优化的方法，用于批量构建具有可控分子拥挤和功能多样性的膜化原细胞模型。我们在膜化原细胞中诱导内源性LLPS的混合-孵育-洗涤方案在微反应器技术、环境传感以及治疗药物的递送和持续释放方面具有潜在应用。
