The organization of DNA into precise structural domains is fundamental to cellular function, yet the physical principles governing this organization at biological membranes remain poorly understood. Here we reveal how membrane dynamics direct the assembly of mitochondrial DNA (mtDNA) nucleoids, essential structures that prevent pathological mtDNA release into the cytosol. Using a synthetic lipid bilayer platform that mimics the inner mitochondrial membrane, we discovered that the mtDNA packaging protein Tfam spontaneously forms liquid condensates through a previously unknown membrane-proximal phase separation mechanism. Multi-modal biophysical analysis revealed that this assembly occurs through prewetting-induced protein condensation near critical membrane interfaces, driven by prewetting protein on the bilayer. Super-resolution microscopy in living cells confirmed that this physical principle is essential for maintaining mtDNA integrity, as disruption of membrane-directed nucleoid assembly triggers mtDNA release. Live-cell super-resolution microscopy and time-resolved fluorescent analysis reveal that these organizational principles are vital for healthy mtDNA maintenance within cells. Disruption of this mechanism leads to mtDNA release, loss of mitochondrial DNA, and activation of the c-GAS/STING pathway, which can be reversibly restored by reestablishing membrane structure. Beyond the distinct biological significance of mitochondrial nucleoid organization, our study generally presents a critical principle regarding how membrane heterogeneity and transient dynamics regulate the generation of membrane-less organelles near the membrane surface.

葛一凡, 本科毕业于南京医科大学, 获药学学士, 博士毕业于普渡大学获化学博士, 于哈佛医学院麻省总医院从事博士后研究, 现任中科院上海有机化学研究所生物与化学交叉中心副研究员。研究方向为膜生物物理, 细胞器动态的体外重构和单分子生物物理分析。