Abstract
Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology.
Highlights
Lipids and proteins are not homogenously distributed in the plasma membrane of eukaryotic cells giving raise to biochemically and biophysically distinct domains
Cells were plated in glassbottom microscope dishes and prior to imaging were washed three times with phosphate buffered saline (PBS) and labeled with PY3304, PY3174 or PY3184 in Dulbecco’s minimum essential medium (DMEM) for 30 min at 5–10 mM
We tested the performance of three new dyes, PY3304, PY3174 and PY3184 as membrane order-sensing probes
Summary
Lipids and proteins are not homogenously distributed in the plasma membrane of eukaryotic cells giving raise to biochemically and biophysically distinct domains. The properties of lipid domains can be investigated by analyzing the localization and dynamics of membrane proteins and lipids [15], most of these approaches rely on prior knowledge of the preference of the probe for partitioning into the ordered and disordered domains. A more direct approach is the use of environmentally sensitive membrane dyes [5,16,17,18,19,20]. These probes typically change their fluorescence properties based on the polarity of their local solvent [21]. Since the degree of lipid packing changes the dyes’ local molecular environment, liquidordered and liquid-disordered membranes can be distinguished by the differential penetration of polar water molecules into the otherwise non-polar bilayer interior
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