Criticality of plasma membrane lipids reflects activation state of macrophage cells

Abstract

Signalling is of particular importance in immune cells, and upstream in the signalling pathway many membrane receptors are functional only as complexes, co-locating with particular lipid species. Work over the last 15 years has shown that plasma membrane lipid composition is close to a critical point of phase separation, with evidence that cells adapt their composition in ways that alter the proximity to this thermodynamic point. Macrophage cells are a key component of the innate immune system, are responsive to infections and regulate the local state of inflammation. We investigate changes in the plasma membrane's proximity to the critical point as a response to stimulation by various pro- and anti-inflammatory agents. Pro-inflammatory (interferon γ, Kdo 2-Lipid A, lipopolysaccharide) perturbations induce an increase in the transition temperature of giant plasma membrane vesicles; anti-inflammatory interleukin 4 has the opposite effect. These changes recapitulate complex plasma membrane composition changes, and are consistent with lipid criticality playing a master regulatory role: being closer to critical conditions increases membrane protein activity.

Keywords: critical lipidomics; liquid–liquid phase separation; macrophage activation; plasma membrane composition.

Figure 1.

The plasma membrane of macrophage cells is close to critical composition and changes its transition temperature in response to signalling molecules. (a) Fluorescence microscope image of GPMVs at 37°C and 3°C. Scale bar, 5 μm. (b–e) Fraction of GPMVs showing just one phase over the total of vesicles observed as a function of the temperature. The data show a sigmoidal trend and are fitted with a hyperbolic tangent from which are extracted the transition temperature at mid-height and the width of the transition. We compare the samples obtained from cells treated with KLA (b), LPS (c) and IFN-γ (e), for 12 h, with a non-treated control condition prepared in parallel. All these ‘pro-inflammatory’ treatments shift the transition temperature towards higher temperatures. The coloured arrow at the bottom indicates the direction of the temperature variation imposed on the GPMV samples during the imaging process. (d) The knock-out TLR4^−/− cells do not vary the transition temperature when stimulated with KLA (in contrast to (a)), remaining the same as the unstimulated controls. (Online version in colour.)

Figure 2.

Anti-inflammatory treatment changes the melting temperature in the opposite direction compared with pro-inflammatory stimuli, consistent with changes in the composition of the membrane away from the critical point. The data show the fraction of uniform GPMVs as the temperature of the sample is varied. The two curves correspond to 24 h of IL-4 stimulation and to unstimulated conditions. T_UNST = (13.11 ± 0.49)°C, T_IL4 = (10.46 ± 0.33)°C. (Online version in colour.)

Figure 3.

Pro- and anti-inflammatory treatments affect the transition temperature systematically. The scatter in the absolute transition temperature (particularly notable in the unstimulated (UNST) cells) is reduced significantly compared with same-day unstimulated controls. (a) Fitted transition temperatures of vesicles produced by macrophage cells treated with IL-4, IFN-γ, LPS or KLA. Each small data marker comes from an experiment with between 300 and 600 vesicles. The large markers indicate the average in each distribution, weighted with the errors on T_m. (b) Temperature difference of each stimulation experiment with its control condition. From one-way analysis of variance, we obtained the distribution differences to be statistically significant with *p < 0.1, **p < 0.05, ***p < 0.005. (Online version in colour.)

Figure 4.

At very low temperatures, irregular-shaped domains are observed and attributed to a gel phase. (a) The fraction of GPMVs with irregular domains (over the total of phase-separated GPMVs) increases at low temperatures. This fraction grows below T_m, as can be seen by comparing in (b) the ‘conventional’ data on liquid–liquid phase separation for the three conditions indicated in the legend. See microscopy images in electronic supplementary material, figure S3. (Online version in colour.)