The Many Layers of Membrane Biophysics: Environment-Sensitive Fluorophores Report on Structural Organization of Biological Membranes at Various Depths

Molecular-order-related membrane biophysical properties contribute to the functional modulation of transmembrane proteins, and therefore, their changes due to a modified membrane composition in diseases associated with alterations in lipid levels may play important pathophysiological roles. In living cells, membrane biophysics can be examined with environment-sensitive fluorophores that describe the structural organization of biological bilayers from different aspects. Microviscosity-sensitive probes such as TMA-DPH characterize the degree of motional freedom, that is, fluidity, polarity-sensitive dyes including Laurdan or PY3174 report on the extent of water penetration, that is, hydration, whereas voltage-sensitive fluorophores such as di-8-ANEPPS quantify the nonrandom alignment of molecular dipoles, that is, the dipole potential. Given that all of the above properties are intrinsically linked to the arrangement of membrane constituents, such parameters are assumed to change in parallel with each other, and thus, the information gained by the environment-sensitive fluorophores is generally considered equivalent. In the current study, using fluorescence-based measurement techniques and manipulating membrane sterol levels with cyclodextrin-based complexes in living cells, we experimentally demonstrate incongruent changes in fluorescence properties of TMA-DPH, Laurdan, PY3174, and di-8-ANEPPS. Comparative MD simulations reveal that this can be due to the distinct membrane localization of the fluorophores. Our experimental and computational analyses reveal that the most commonly applied environment-sensitive fluorophores depict the structural organization of membranes at different depths and suggest that a comprehensive investigation of bilayer structure should include an appropriate combination of dyes, which would be required for a better understanding of membrane biophysics and its potential roles in disease pathogenesis.