Viscosity-Sensitive Membrane Dyes as Tools To Estimate the Crystalline Structure of Lipid Bilayers

Lipid membranes are crucial for cellular integrity andregulation,and tight control of their structural and mechanical properties isvital to ensure that they function properly. Fluorescent probes sensitiveto the membrane's microenvironment are useful for investigatinglipid membrane properties; however, there is currently a lack of quantitativecorrelation between the exact parameters of lipid organization anda readout from these dyes. Here, we investigate this relationshipfor molecular rotors, or microviscosity sensors, bysimultaneously measuring their fluorescence lifetime to determinethe membrane viscosity, while using X-ray diffraction to determinethe membrane's structural properties. Our results reveal aphase-dependent correlation between the membrane's structuralparameters and mechanical properties measured by a BODIPY-based molecularrotor, giving excellent predictive power for the structural descriptorsof the lipid bilayer. We also demonstrate that differences in membranethickness between different lipid phases are not a prerequisite forthe formation of lipid microdomains and that this requirement canbe disrupted by the presence of line-active molecules. Our resultsunderpin the use of membrane-sensitive dyes as reporters of the structureof lipid membranes.

Automated summary

Lipid membranes play a crucial role in cellular integrity and regulation, and their structural and mechanical properties need to be tightly controlled for proper function. In this study, we investigated the correlation between lipid organization parameters and a molecular rotor's fluorescence readout by simultaneously measuring fluorescence lifetime and using X-ray diffraction. Our results showed a phase-dependent correlation between the structural parameters and mechanical properties of lipid bilayers, providing accurate prediction of the lipid membrane's structure. Moreover, we found that membrane thickness differences between lipid phases are not necessary for the formation of lipid microdomains and can be disrupted by the presence of line-active molecules. These findings support the use of membrane-sensitive dyes as reporters of lipid membrane structure.