Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis
| Autor: | Thorsten Wohland, Andreas Herrmann, Thomas Korte, Anjali Gupta |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Fluorescence-lifetime imaging microscopy Liquid ordered phase liquid disorder Membrane biology liquid order QD415-436 030204 cardiovascular system & hematology Biochemistry Cell Line 03 medical and health sciences 0302 clinical medicine Endocrinology Microscopy Methods Animals Unilamellar Liposomes Total internal reflection fluorescence microscope Molecular Structure Chemistry diffusion membrane phase Cell Membrane Cell Biology Lipids Fluorescence Spectrometry Fluorescence 030104 developmental biology Membrane Microscopy Fluorescence Biophysics lipids (amino acids peptides and proteins) Sphingomyelin |
| Zdroj: | J Lipid Res Journal of Lipid Research, Vol 61, Iss 2, Pp 252-266 (2020) |
| ISSN: | 0022-2275 |
| DOI: | 10.1194/jlr.d119000364 |
| Popis: | A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines. |
| Databáze: | OpenAIRE |
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