Detergent-Triggered Membrane Remodelling Monitored via Intramembrane Fluorescence De-Quenching

Detergent-induced membrane solubilization is important for several biotechnological applications including membrane protein isolation, cell lysis and virus inactivation. The thermodynamic details of the underlying process have been previously examined, but the mechanistic details remain largely underexplored owing in part, to a lack of suitable technologies capable of assessing nanoscopic membrane disruption events. Key open questions include: how do detergents remodel the membrane structure at sub-solubilizing concentrations? And what is the sequence of morphological transitions that lead up to solubilization? Here, we introduce a single-colour assay based on the fluorescence de-quenching of membrane-integrated fluorophores as a sensitive and generalizable tool to probe nanoscale membrane remodelling events induced by detergents. We demonstrate, using fluorescence spectroscopy and time-correlated single photon counting, that the widely used detergent Triton X-100 triggers substantial morphological changes at concentrations below its critical micellar concentration. Moreover, by taking advantage of single vesicle fluorescence lifetime imaging and scanning electron microscopy, we reveal that the swelling step involves a morphological transition from spherical vesicles to toroidal structures, providing direct evidence for detergent-driven membrane reorganization prior to solubilization. Our findings support and refine a multistep model of detergent-induced membrane solubilization, positioning fluorescence de-quenching as a tool for detecting conformational intermediates. We show that the fluorescence de-quenching approach performs robustly across multiple cyanine-based probes and experimental conditions and its nanoscale sensitivity provides a platform from which to interrogate membrane perturbations induced by a wide variety of molecular disruptors, including those with important biomedical significance.