Fluorescence Lifetime Imaging of Membrane Lipid Order with a Ratiometric Fluorescent Probe

Abstract

To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N(∗)) and tautomer (T(∗)) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T(∗) form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N(∗) form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis.

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion

“…We loaded our cells with FliptR and determined the effects of acute glycolysis inhibition on membrane order in TZM-bl cells and compared these effects to cells supplemented with or depleted of cholesterol ( Fig 5B). The distribution of lifetimes was homogenous throughout the entirety of the cell regardless of cholesterol enrichment or depletion ( Fig 5A and 5B), indicating that the liquid-ordered and disordered phases are mixed in our spatiotemporal resolution, which has been reported previously in similar time-correlated single photon counting (TCSPC) platforms [52,55]. Treatment with 1mM MBCD showed reductions in lifetime by more than 200ps ( Fig 5B), indicating a drop in membrane order.…”

Liquid-Ordered Phase Formation by Mammalian and Yeast Sterols: A Common Feature With Organizational Differences

“…Recent studies in both yeast and mammalian cells have challenged the hypothesis that the main role of sterols is solely to generate l d /l o lateral membrane heterogeneity (Souza et al, 2011). FLIM results and the high sterol content of eukaryotic plasma membranes suggest that an l o -like phase may actually correspond to a major area of the cell membrane (Owen et al, 2006;De Almeida and Joly, 2014;Kilin et al, 2015). These diverse perspectives can be reconciled by hypothesizing that while not all sterols are able to form l o phase, the ability to form such phases in membrane model systems seems to be a common underlying feature of the main sterols of eukaryotic organisms.…”

PTEN activation contributes to neuronal and synaptic engulfment by microglia in tauopathy

“…During apoptosis, plasma membranes lose their molecular asymmetry through a mechanism by which PS, a negatively charged amine-containing phospholipid, becomes externalized to the outer leaflet of the cell membrane [34]. We therefore sought to determine whether PS is exposed outside of the cell membrane in tauopathy by probing rTg4510 and WT primary neurons with the membrane asymmetry probe F2N12S, which responds to changes in the charge of the membrane following PS exposure [9,11,20,25,49]. Live imaging of rTg4510 compared to WT primary neurons labeled with F2N12S revealed a pronounced gain in membrane symmetry, indicating increased exposure of PS in the presence of pathological Tau (Fig.…”