Formation and function of lipid nano-domains investigated through model membrane systems

Abstract

The plasma membrane plays a fundamental role in cellular biology, separating the cell from its surroundings and regulating key processes such as macromolecular trafficking, cell-cell communication, and signalling. However, the plasma membrane is a highly complex, heterogeneous, and dynamical structure which renders its study often challenging. As a result, model membrane systems have been developed, offering simplified biomimetic platforms to study membrane phenomena under controlled conditions and with various experimental techniques.
This thesis aims to provide new insights into the structure and function of the plasma membrane, with a particular focus on the formation and role of nanoscale lipid domains.
Chapter 1 introduces plasma membranes, both from a functional biological perspective and from a biophysical point of view, highlighting general features of structure, function and behaviour, including the membrane thermodynamics. The chapter concludes with a discussion outlining the gaps in understanding that the thesis proposes to answer.
Chapter 2 describes the different techniques used throughout the thesis, namely a combination of atomic force microscopy (AFM) and calorimetric analysis. AFM offers molecular-level details and localised mechanical characterisation while calorimetry provides a bulk thermodynamic quantification, making it possible to compare and contrast the behaviour of supported and unsupported membranes.
In Chapter 3, a novel model membrane systems for Escherichia coli inner membrane is developed, aiming to accurately replicate the compositional, thermodynamical, and mechanical properties of the native lipid membrane. Given the importance of E. coli as a bacterial model, the results offer a new platform for in vitro studies, particularly where involving membrane proteins and mechanotransduction.
Chapter 4 investigate the impact of supporting the bilayer on a solid, showing that the membrane phase behaviour is significantly altered by surface interactions over multiple timescales. At room temperature, the E. coli model membrane developed in Chapter 3 undergoes multiple complex reorganisation consistent with a surface-induced bias in the lipid flip-flop, enabling local compositional fluctuations and long-term leaflet asymmetry.
Chapter 5, the last experimental chapter, explores the role of lipid phases in extracellular vesicle (EV) uptake by eukaryotic membranes, demonstrating that phase-separated domains are central to EV uptake. Even in the absence of membrane proteins, EVs primarily interact with ordered, less fluid lipid domains, highlighting the functional role of lipid rafts in native membranes. Observed variations in EV-membrane fusion mechanisms, depending on EV origin, offer additional insights into a process that has been widely debated.
Finally, Chapter 6 discusses all the results together, aiming to address some of the questions raised in the introduction and where relevant suggest future experiments and developments in the field.