Elettra | NanoInnovationLab Lipid phase behaviour in plasma membranes: an overlooked determinant of membrane function When molecules approach a cell, they face a selective barrier, known as plasma membrane, which regulates their access into the cell. The plasma membrane is a complex structure made up of thousands of lipids and proteins that self-assemble to form a double-layered matrix. This matrix consists of unevenly arranged lipids with embedded proteins. The distribution of the components in functional domains, as well as their distribution in the two membrane leaflets, varies significantly depending on the specific type of cell, its pathological state, and the surrounding environmental conditions. The functionality of membrane is highly dependent on its local macromolecular composition and assembling which in turn influence the chemical and mechanical properties of the membrane itself as well as the way the membrane responds to external stimuli, regulating processes at the level of the entire cell, as cell migration and intracellular signalling. Importantly, the fate of molecules that encounter the membrane barrier is determined by the specific characteristics of the interaction site. Providing the tools to investigate the molecular mechanisms that govern the distribution of membrane components in relation to cell behaviour, would allow to uncover the molecular mechanisms underlying the development and spread of diseases. Additionally, it would strongly contribute to study how drugs and drug carriers interact with cell, allowing to optimize their design to improve their effectiveness towards personalized medicine routes. One effective strategy for addressing the intrinsic complexity of biological membranes is to use advanced physical techniques on simplified systems. These systems consist of a limited number of molecules in controlled molar proportions, which mimic key biological features of the membrane domain of interest. Currently, simplified membrane models that replicate specific membrane domains are not comprehensive. Their development is constrained by the availability of purified molecules, particularly proteins, and the challenges of synthesizing systems with a defined geometry, as flat membranes supported by solid substrates. These factors limit accessibility for investigation using advanced techniques. In this context, we focused on the development of a suitable model for "solid rafts", membrane domains primarily composed of saturated lipids. They are referred to as "solid" because they exhibit high rigidity compared to the surrounding membrane environment. These self-forming, persistent domains play a crucial role in intracellular transport, inter-organellar communication, and lipid transfer. Our membrane model containing solid raft mimics was composed by the phospholipid DMPC, the sphingolipid sphingomyelin, and cholesterol, mixed in a molar ratio of 2:1:0.15. In this lipid mixture, rigid solid-ordered (So) domains coexist with more fluid liquid-disordered (Lα) phase-separated domains, as a consequence of liquid–liquid separation of lipids, driven by preferential interactions. This is expected to occur in mixtures of lipids with different transition temperature, as DMPC melting around 24°C and sphingomyelin, known to melt at 35°C, cholesterol acting as a phaseseparation promoter. Using complementary physical techniques, our study offers a comprehensive understanding of the features and lipid organisation in this biomimetic model. DMPC was selected as phospholipid matrix due to its availability in deuterated form, which enables neutron-based studies, and its accessible transition temperature, facilitating calorimetric (DSC) and atomic force microscopy (AFM) analyses. For the first time, AFM measurements conducted at the NanoInnovation Lab of Elettra allowed us to visualize the realtime formation of lipid raft So domains in a ternary mixture during temperature increase. This revealed distinct morphological structures both below and above the transition temperature. AFM imaging of membrane morphology during the phase transition, shown in Fig. 2, highlighted the complex structure of the supported raft at low temperatures, when existing in the gel phase. This gel phase was characterized by grooves and indentations that were found to guide the assembly of the solid ordered domains within the surrounding disordered fluid phase upon the increase in temperature. The supported lipid bilayer exhibited reproducibility, rapid deposition kinetics on supports, showing high symmetry between its two leaflets, as demonstrated by neutron reflectometry (Fig. 3). This symmetry is crucial for the validity of the model, as it shows that all the three molecular constituents are exposed on the outer surface of the supported membrane, creating a unique interface accessible to the incoming macromolecules. In addition, Differential Scanning Calorimetry (DSC) analysis (Fig 4) revealed that sphingomyelin increases the transition temperature of the phospholipid matrix. This indicates that stronger intramolecular bonds form between sphingomyelin and DMPC phospholipid, thereby adding rigidity to the membrane. Additionally, sphingomyelin broadens the phase transition process, providing enhanced stability to the raft complex. Conversely, cholesterol promotes phase separation, as evidenced by the emergence of new transition peaks in the DSC thermograms of binary and ternary mixtures containing it. Our findings underscore the importance of including cholesterol in membrane raft models. This study advances the field of membrane models and lipid raft research by enabling the mimicry of solid membrane domains and opening the way for a variety of future studies exploring their functionality as a function of the surrounding environment, external stimuli and the presence of incoming biomacromolecules. Elettra Highlights 2024–2025 46
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