Lipid-Conjugated Rigidochromic Probe Discloses Membrane Alteration in Model Cells of Krabbe Disease

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.

Reduction in miR‐219 expression underlies cellular pathogenesis of oligodendrocytes in a mouse model of Krabbe disease

“…In the present study, we have shown that miR-219 reduced psychosine accumulation in developing twitcher OLs (Figure 5). Recent studies have shown that psychosine preferentially accumulates in membrane microdomains in the brain of KD patients and twitcher mice, leading to the disruption of lipid rafts that play a crucial role in membrane trafficking and cell signaling (44)(45)(46)(47). In addition, it has been known that psychosine mediates apoptotic cell death via caspase-3 activation in various cell types (45,(48)(49)(50).…”

Molecular Rotors: Fluorescent Sensors for Microviscosity and Conformation of Biomolecules

“… [70] Although not covered here in detail, other rotor dyes have also been used to investigate the microviscosity of lipid membranes and intracellular compartments; through intensity, ratiometric, or lifetime‐based measurements. These include merocyanine, [112] anthracene, [113] triphenylamine, [114] juliodine,[ 32 , 33 ] porphyrin,[ 45a , 46b ] pyrrolic compounds, [115] GFP, [87a] or indocarbocyanine nanoparticle [116] —based molecular rotors.…”

FLUTE: A Python GUI for interactive phasor analysis of FLIM data

“…The main advantages of phasor analysis are that it provides a visual distribution of the molecular species by clustering pixels with similar decays, it allows for color mapping of the pixels of the FLIM images, and it is linear in terms of non-interacting molecular species. (15,16,21,22) Phasor analysis of FLIM data is increasingly used in several fields of biomedical research (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) and is particularly useful for metabolic imaging with the endogenous biomarkers NAD(P)H and FAD to map their complex autofluorescence distributions in live tissues in the context of different physiopathological processes. (5,16, .…”