Real-Time Confocal Imaging of Rare Morphological Transitions in Protein-free GUVs Uncovers Shallow Energy Landscapes for Membrane Assemblies

Lipid vesicles exhibit a rich variety of morphologies shaped by the interplay of composition, curvature, and mechanical perturbations. In this study, we study rare spontaneous morphological remodeling events in phase separating (POPC:DPPC) and non-phase separating (POPC:DOPE) protein-free giant unilamellar vesicles (GUVs), under apparently unperturbed (isothermal/physical and chemical) conditions using real-time confocal microscopy. Quantitative analysis of these events through geometric analysis and Canham-Helfrich elastic energy modeling revealed two distinct mechanistic classes of morphological transformations: those with stable elastic energy profiles (occurring over shallow energy landscapes) and, energetically dissipative relaxations (after possible transient mechanical perturbations from sample handling). The study highlights how lipid composition alone can endow membranes with dynamic shape plasticity, underscoring the role of curvature-composition coupling in accessing metastable morphologies, thereby allowing kinetic windows for stabilization of specific morphologies with compositional interventions. These findings offer new insight into how membranes may respond to subtle environmental cues and how early prebiotic membranes or protein-free compartments might have sampled and stabilized complex shapes in cellular evolution.