Abstract
The plasma membrane of cells has a complex architecture based on the bidimensional liquid-crystalline bilayer arrangement of phospho- and sphingolipids, which in turn embeds several proteins and is connected to the cytoskeleton. Several studies highlight the spatial membrane organization into more ordered (L o or lipid raft) and more disordered (L d ) domains. We here report on a fluorescent analog of the green fluorescent protein chromophore that, when conjugated to a phospholipid, enables the quantification of the L o and L d domains in living cells on account of its large fluorescence lifetime variation in the two phases. The domain composition is straightforwardly obtained by the phasor approach to confocal fluorescence lifetime imaging, a graphical method that does not require global fitting of the fluorescence decay in every spatial position of the sample. Our imaging strategy was applied to recover the domain composition in human oligodendrocytes at rest and under treatment with galactosylsphingosine (psychosine). Exogenous psychosine administration recapitulates many of the molecular fingerprints of a severe neurological disease, globoid cell leukodystrophy, better known as Krabbe disease. We found out that psychosine progressively destabilizes plasma membrane, as witnessed by a shrinking of the L o fraction. The unchanged levels of galactosyl ceramidase, i.e., the enzyme lacking in Krabbe disease, upon psychosine treatment suggest that psychosine alters the plasma membrane structure by direct physical effect, as also recently demonstrated in model membranes.
Original language | English |
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Pages (from-to) | 477-486 |
Number of pages | 10 |
Journal | Biophysical Journal |
Volume | 116 |
Issue number | 3 |
Early online date | 25 Dec 2018 |
DOIs | |
Publication status | Published - 5 Feb 2019 |
Funding
The authors acknowledge Prof. Piet Dijkstra (Twente University) for useful discussions. This research was supported by 1) Regione Toscana, Bando Fondo Aree Sottoutilizzate Salute 2014 , under the framework of the project “DIAMANTE-Diagnostica Molecolare Innovativa per la scelta terapeutica personalizzata dell’adenocarcinoma pancreatico” (grant number CUP I56D15000310005 ); 2) Fondazione Cassa Di Risparmio di Lucca , under the framework of the project “Pre-Clinical Testing of Lithium Treatment in Krabbe Disease”; 3) European Leukodystrophy Association (ELA) International , under the framework of the project “Development of a novel, nanovector-mediated enzyme replacement therapy for Globoid Cell Leukodystrophy (GLD)”, grant no. ELA 2015-010C1A .