Imaging dynamic pore-formation processes in droplet-interface bilayers

Observing dynamic pore-forming processes in lipid bilayers is a challenging task. It requires a bilayer system that is stable, compositionally adjustable, and one that may be interrogated using electrical and optical techniques. Aside from these practical challenges, and that pore formation is inherently destructive to the bilayer system, membrane pores are often highly transient, with milli- or microsecond lifetimes, and are seldom larger than a few tens of nanometres in diameter. This work presents droplet-interface bilayers (DIBs) as a platform for overcoming some of these challenges. The technique is in particular applied to the study of electroporation, a well-known and widely used but less comprehensively understood phenomenon in which transmembrane pores are generated under the influence of an applied electric field. DIBs are shown to be a useful system for the study of the formation, development, energetics and dynamics of electropores. Through simultaneous electrophysiological recording and calcium-flux imaging, the real-time behaviour of individual electropores within an ensemble of transmembrane defects is observed for the first time. This reveals information about the pore dynamics, their diffusion in the membrane, and their gating kinetics. Analysis of these kinetics further allows the determination of the energy barriers to pore formation. This is the first such imaging study to be carried out on electroporation, granting access to electropore behaviour that previously no imaging methodologies have accessed. In turn, this paves the way for imaging studies of more complex pore-forming processes, such as those mediated by pore-forming peptides and proteins. Work on these systems shall be presented towards the end of this thesis, in particular, an investigation into the interactions of four typical antimicrobial peptides (AMPs) with DIBs, which serves to expand the toolkit for membrane-active peptide studies. In this work, for the first time, the location of dynamic membrane pores is correlated with the position of the pore-forming peptides by combining calcium-flux and single-molecule imaging, revealing real-time information about peptide-mediated pores that is difficult to access through alternative bilayer membrane methods.</p