Plant sterol clustering correlates with membrane microdomains as revealed by optical and computational microscopy

Abstract

Local inhomogeneities in lipid composition play a crucial role in the regulation of signal transduction and membrane traffic. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phyto-sphingolipids. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used a new push–pull pyrene probe and fluorescence lifetime imaging microscopy (FLIM) combined with all-atom mul-tiscale molecular dynamics simulations to provide a detailed view on the interaction between phos-pholipids and phytosterol and the effect of modulating cellular phytosterols on membrane-associ-ated microdomains and phase separation formation. Our understanding of the organization princi-ples of biomembranes is limited mainly by the challenge to measure distributions and interactions of lipids and proteins within the complex environment of living cells. Comparing phospholip-ids/phytosterol compositions typical of liquid-disordered (Ld) and liquid-ordered (Lo) domains, we furthermore show that phytosterols play crucial roles in membrane homeostasis. The simulation work highlights how state-of-the-art modeling alleviates some of the prior concerns and how unre-futed discoveries can be made through a computational microscope. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of plant cells and suggest that they are key compounds for the formation of plant PM microdomains and the lipid-ordered phase.