Giant unilamellar vesicles (GUVs) serve as a well-defined model system for investigating the physical principles underlying synthetic cell division. A central challenge is to direct GUVs toward division-compatible morphologies, for example dumbbell-shaped intermediates with a distinct neck, while avoiding non-productive deformations such as elongated tubes or deep invaginations. Although membrane deformation and volume confinement are known to act synergistically in cell division, how spontaneous curvature and reduced volume jointly determine whether a vesicle enters division-related deformation pathways remains experimentally unexplored.
In this work, we systematically investigate this synergy using a low-perturbation microfluidic perfusion platform combined with confocal imaging. Membrane spontaneous curvature is independently tuned by incorporating different concentrations of lysophosphatidylcholine (LPC) into a POPC/POPE base membrane, while transmembrane osmotic pressure differences are applied by varying the external glucose concentration to precisely control the reduced volume. Real-time imaging reveals that the sign of spontaneous curvature dictates the deformation direction: positive curvature drives an outward deformation pattern; whereas negative curvature induces an inward deformation pattern. Increasing the osmotic pressure difference promotes deeper deformation by decreasing the reduced volume, while the curvature sign determines whether the deformation proceeds outward or inward.
By constructing a morphology diagram with LPC concentration and osmotic pressure difference as coordinates, we identify six distinct morphological regimes. Importantly, the division-compatible dumbbell morphology with a well-defined neck emerges only within an intermediate window of both positive spontaneous curvature and moderate volume shrinkage. Excessive curvature or osmotic stress redirects the system into non-productive long-tail or deep-invagination states. The elongated tail represents a highly localized nonlinear budding instability that lies beyond the scope of linear stability analysis. This study reveals the synergistic regulation of vesicle deformation by spontaneous curvature and reduced volume, providing an experimental basis for the rational design of divisible artificial cells.