Protein-free membrane fusion: a refined view of the delicate fusogenic properties of calcium

In the late 20th century, calcium took on the identity of an independent fusogen, when it was found to induce fusion of anionic large unilamellar vesicles (LUVs), yet its ability to drive fusion in cell-sized membranes remains poorly understood. Here, we directly quantify calcium-mediated fusion of giant unilamellar vesicles (GUVs) using a microfluidic trapping platform combined with confocal microscopy, enabling simultaneous measurement of lipid mixing, content mixing, and fusion outcomes across hundreds of single vesicles. We systematically map fusion efficiency as a function of calcium concentration, membrane composition, and mechanically imposed tension. We find that calcium-induced fusion of GUVs in the absence of proteins is remarkably fickle and composition-sensitive, as the vesicles need to be sufficiently instable to allow the opening of the fusion pore, yet stable enough to prevent bursting and collapse. Negatively charged GUVs containing high fractions of DOPE exhibit the highest fusogenic responsiveness, whereas other compositions undergo extensive lipid mixing without pore formation. Increasing membrane tension can shift this balance and promote full fusion, revealing a narrow parameter space in which calcium acts as an effective protein-free fusogen for cell-sized membranes. These findings clarify longstanding discrepancies between LUV- and GUV-based calcium fusion assays and provide quantitative design rules for employing calcium as a fusogen in synthetic biology and membrane-reconstitution studies, where controlled membrane growth, vesicle-vesicle fusion, and module integration are central to building and sustaining artificial cells. SIGNIFICANCECalcium is widely regarded as a potent, protein-free fusogen in nanometer-scale lipid vesicles, yet its relevance for fusion between cell-sized membranes remains unresolved. This gap limits our ability to translate decades of LUV-based fusion studies to more physiologically realistic systems and restricts the use of calcium in synthetic cell engineering. By directly quantifying calcium-mediated fusion in giant unilamellar vesicles (GUVs), we identify the membrane compositions and mechanical conditions under which calcium can reliably drive full fusion rather than mere lipid mixing. Our findings provide the first systematic map of calciums fusogenic parameter space in cell-sized membranes, enabling more informed design of fusion-based assays, reconstitution experiments, and strategies for membrane growth in artificial cells.