The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed. Polarity-sensitive dyes are powerful tools to characterize such lipid membrane order, employing, for example, confocal and two-photon microscopy. The investigation of potential nanodomains, however, requires the use of superresolution microscopy. Here, we test the performance of the polarity-sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA, and NR12S in superresolution stimulated emission depletion microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles, show the high potential of these dyes for probing nanoscale membrane heterogeneity.
The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.
The freshwater algae Ochramonas danica produces a range of polychlorinated single-chain amphiphiles. Danicalipin A, a hexachlorosulfolipid makes up 90% of the polar lipid content of the flagellar membrane of O. danica. Its exotic structure presents both synthetic challenges and raises questions about its function within the membrane. To address these questions, we have combined total synthesis and membrane biophysics to investigate the effects of structural elements of chlorosulfolipids on their behavior in monolayers and bilayers. The discovery of a titanium-based catalytic, enantioselective dichlorination of allylic alcohols enabled the eight-step synthesis of (þ)-Danicalipin A as a single stereoisomer in sufficient quantities for in vitro analysis. Nanoscale secondary ion mass spectrometry (NanoSIMS) confirmed that Danicalipin A is localized within plasma membrane of O. danica cells. Preliminary biophysical characterization of Danicalipin A has revealed that it alters the phase behavior and lateral organization in monolayers and bilayers of other lipids present in the membrane of O. danica. Danicalipin A incorporates into monolayers of phospho-and glycolipids at the air-water interface and increases the surface pressure at which the liquid-expanded to liquid-compact phase transition occurs, as well as increasing monolayer compressibility. Similarly, in giant unilamellar vesicles, Danicalipin A lowers the transition temperature of saturated phosphoand glycerolipids and causes phase separation. Natural and unnatural analogs to Danicalipin A are being synthesized to examine the effects of the stereochemistry, chlorination pattern, and sulfation on these biophysical properties. These results may reveal how the membrane of O. danica accommodates high concentrations of chlorosulfolipids, which are toxic to other organisms. Mapping water molecules across lipid bilayers at high spatial resolution is important for both understanding membrane biophysics and membrane protein biological function. Current biophysical methods to determine water concentration in biological systems have several limitations, the main one being insufficient spatial resolution. Water penetration profiles of lipid bilayers have been measured using electron spin echo envelope modulation (ESEEM) of nitroxide spin labels based on magnetic interactions of nitroxides with the matrix water molecules. However, the matrix ESEEM effect is caused by through-space dipolar interactions, restricting spatial sensitivity to 10 Å , a distance which spans a significant portion of the lipid bilayer. Overhauser DNP is a less direct method to determine local water concentration, via measuring bulk water polarization induced by microwave irradiation of a spin label, and depends on complex spin dynamics inside the lipid bilayer. Hyperfine sublevel correlation (HYSCORE) spectroscopy is a sensitive technique to detect hydrogen bonds formed with paramagnetic centers. Here we demonstrate the use of HYSCORE spectroscopy to directly and accurately measure the fraction of wa...
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