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.

Signalling is of particular importance in immune cells, and upstream in the signalling 11 pathway many membrane receptors are functional only as complexes, co-locating with particular 12 lipid species. Work over the last 15 years has shown that plasma membrane lipid composition is 13 close to a critical point of phase separation, with evidence that cells adapt their composition in 14 ways that alter the proximity to this thermodynamical point. Macrophage cells are a key 15 component of the innate immune system, responsive to infections, regulating the local state of 16 inflammation. We investigate changes in the plasma membrane's proximity to the critical point, as 17 a response to stimulation by various pro-and anti-inflammatory agents. Pro-inflammatory (IFN-, 18 Kdo-LipidA, LPS) perturbations induce an increase in the transition temperature of the GMPVs; 19 anti-inflammatory IL4 has the opposite effect. These changes recapitulate complex plasma 20 membrane composition changes, and are consistent with lipid criticality playing a master 21 regulatory role: being closer to critical conditions increases membrane protein activity. 22 23 129 GPMV buffer. GPMV buffer is formed by 10mM HEPES, 150mM NaCl, 2mMCaCl 2 , the pH is adjusted 130 to 7.4 with HCl or NaOH. Lastly the vesiculating agent is added and the cells are left in the incubator 131 for 1.5 hours at 37 • C. 20 l of vesiculating agent (2mM DTT, 25mM PFA) is used for each ml of GPMV 132 buffer. The medium is gently harvested and transferred into a falcon tube. The sample is left at 133 37 • C enough to let the blebs deposit on the bottom of the tube: for a volume of 4 ml, 24 hours are 134 enough for the whole sample to sediment. 135 4 of 15 377 Research was funded by EU Marie Curie action ITN TransPol (EC), NIH-R01GM110052 and NSF-378 MCB1552439 (SLV), Cambridge University Commonwealth, European and International Trust (JS) 379 and ITN BioPol (PC), Wellcome Trust Investigator grant 08045/Z/15/Z (CEB). of lipid phases in 431 model and plasma membranes.