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

Lipid membranes are crucial for cellular integrity and regulation, and tight control of their structural and mechanical properties is vital to ensure that they function properly. Fluorescent probes sensitive to the membrane's microenvironment are useful for investigating lipid membrane properties; however, there is currently a lack of quantitative correlation between the exact parameters of lipid organization and a readout from these dyes. Here, we investigate this relationship for "molecular rotors", or microviscosity sensors, by simultaneously measuring their fluorescence lifetime to determine the membrane viscosity, while using X-ray diffraction to determine the membrane's structural properties. Our results reveal a phase-dependent correlation between the membrane's structural parameters and mechanical properties measured by a BODIPY-based molecular rotor, giving excellent predictive power for the structural descriptors of the lipid bilayer. We also demonstrate that differences in membrane thickness between different lipid phases are not a prerequisite for the formation of lipid microdomains and that this requirement can be disrupted by the presence of line-active molecules. Our results underpin the use of membrane-sensitive dyes as reporters of the structure of lipid membranes.

Measuring the Viscosity of the Escherichia coli Plasma Membrane Using Molecular Rotors.

The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5-40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes.

Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors.

In order to fully understand the dynamics of processes within biological lipid membranes, it is necessary to possess an intimate knowledge of the physical state and ordering of lipids within the membrane. Here we report the use of three molecular rotors based on meso-substituted boron-dipyrrin (BODIPY) in combination with fluorescence lifetime spectroscopy to investigate the viscosity and phase behaviour of model lipid bilayers. In phase-separated giant unilamellar vesicles, we visualise both liquid-ordered (Lo) and liquid-disordered (Ld) phases using fluorescence lifetime imaging microscopy (FLIM), determining their associated viscosity values, and investigate the effect of composition on the viscosity of these phases. Additionally, we use molecular dynamics simulations to investigate the orientation of the BODIPY probes within the bilayer, as well as using molecular dynamics simulations and fluorescence correlation spectroscopy (FCS) to compare diffusion coefficients with those predicted from the fluorescence lifetimes of the probes.

Visualising the membrane viscosity of porcine eye lens cells using molecular rotors.

The plasma membranes of cells within the eye lens play an important role in metabolite transport within the avascular tissue of the lens, maintaining its transparency over the entire lifespan of an individual. Here we use viscosity-sensitive 'molecular rotors' to map the microscopic viscosity within these unusual cell membranes, establishing that they are characterised by an unprecedentedly high degree of lipid organisation.

Imaging plasma membrane phase behaviour in live cells using a thiophene-based molecular rotor.

Molecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscosity-sensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.