Laurdan Monitors Different Lipids Content in Eukaryotic Membrane During Embryonic Neural Development

Abstract

We describe a method based on fluorescence lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stage of development (day 12 (E12) and day 16 (E16) of gestation). For the FLIM measurements, we use the Laurdan probe which is commonly used to asses membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach we build a fluidity scale based on calibration with model systems of different lipid composition. In neuronal cells we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cells groups, E12 and E16. Comparison with NIH3T3 cells show that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.

Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

“…Using phasor analysis Bonaventura et al (2013) have shown the existence of regions of different membrane’s fluidity in E6 and E12 neuronal precursor cells. This is probably due to the difference in their lipid composition which fluctuates laterally provoking the average membrane fluidity to be similar at the two indicated time points [ 45 ]. Researchers showed that cholesterol modifies phospholipid bilayer phase domain properties, its water concentration and dynamics [ 46 , 47 ].…”

Defining Lineage-Specific Membrane Fluidity Signatures that Regulate Adhesion Kinetics

“…Both studies indicate that membrane fluidity can be the more generic parameters influencing biological function of cells over adhesion characteristics. Given the important role of lipid membranes (i.e., membrane fluidity) in biological function such as cell adhesion, previous studies have characterized the fluidic properties of human primary neurons ( Bonaventura et al., 2014 , Noutsi et al., 2016 ), while others have examined the lipidomic profiles of lysed hepatocyte-like cells ( Kiamehr et al., 2017 ). However, limited numbers of studies have been performed to address its cell-type-specific signatures, especially in the context of stem cell biology; thus, the comprehensive characterization of membrane fluidity in various lineages is essential for gaining key membrane insights into cellular identity and differentiation.…”

LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity

“…60 As stated earlier, the fluorescent properties of LAURDAN may change due to the polarity of the environment and also due to dipolar relaxation of water molecules around the LAURDAN dipole during the emission lifetime. [61][62][63][64] Independent phasor analysis of two simultaneous FLIM images, obtained through different bandpass filters, allows the evaluation of these two properties separately. The blue channel is used to evaluate the polarity, and the green channel to observe dipolar relaxation (DR).…”

“…The blue channel is used to evaluate the polarity, and the green channel to observe dipolar relaxation (DR). 60,62,65,66 By monitoring the decay through the two bandpass filters (the GP filters), we can isolate LAURDAN information from different environments: the blue channel refers to LAURDAN where no relaxation occurs and the green channel where DR take place. Figure 5 shows an example of this methodology to study the effect of H 2 O 2 in NIH-3T3 cells.…”

“…Application of the FLIM phasor approach to LAURDAN fluorescence in membranes of live cells was introduced by Golfetto, Hinde, and Gratton in 2013 . As stated earlier, the fluorescent properties of LAURDAN may change due to the polarity of the environment and also due to dipolar relaxation of water molecules around the LAURDAN dipole during the emission lifetime. − Independent phasor analysis of two simultaneous FLIM images, obtained through different bandpass filters, allows the evaluation of these two properties separately. The blue channel is used to evaluate the polarity, and the green channel to observe dipolar relaxation (DR). ,,, By monitoring the decay through the two bandpass filters (the GP filters), we can isolate LAURDAN information from different environments: the blue channel refers to LAURDAN where no relaxation occurs and the green channel where DR take place.…”