Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerization decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of phosphatidylinositol 4,5-bisphosphate lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, leads to the formation of ordered domains that is correlated with an increase in cell peripheral actin filaments. In membrane blebs, which are detached from the underlying actin filaments the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form where actin filaments attach to the plasma membrane via phosphatidylinositol 4,5-bisphosphate. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca²⁺. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.

T cell signaling emanates from large lipid raft platforms. Whether lipid rafts form upon T cell receptor (TCR) engagement or exist in resting T cells was the focus of this study. Plasma membrane order was followed in live T cells at 37°C using laurdan to report on lipid packing. Patching of the TCR in both Jurkat and human primary CD4+ T cells resulted in higher fractions of ordered plasma domains in the patches but did not increase the overall membrane order. The TCR colocalized with actin filaments in unstimulated Jurkat T cells and this colocalization was most prominent for cells in G1 phase. Moreover, the TCR located to the nuclear envelope, in addition to the plasma membrane, in cells in S and G2/M phase. Our study suggests that the TCR resides in ordered plasma membrane domains/lipid rafts that are linked to actin filament and aggregate upon T cell activation.