Fluorescence anisotropy based single liposome assay to measure molecule-membrane interactions.

Nanometer-scaled liposomes are used frequently for research, therapeutic, and analytical applications as carriers for water-soluble molecules. Recent technical advances allow the monitoring of single liposomes, which provides information on heterogeneous properties that were otherwise hidden due to ensemble averaging. Recent observations demonstrated that the efficiency of entrapping water-soluble molecules increases with decreasing vesicle size. The molecular mechanism behind this observation is not clear, but enhanced molecule-membrane interactions due to the increase of the surface area-to-volume ratio could play an important role. To investigate this hypothesis, we extended our single liposome assay based on confocal fluorescence imaging by implementation of fluorescence anisotropy. This combination has not been widely exploited, and confocal fluorescence anisotropy imaging in particular has seldom been used. We investigated different small dye molecules and were able to determine if these molecules interact or not with the liposome membrane. We confirm the liposome size-dependent entrapment of molecules whereas the molecule-membrane interactions appear to be independent of liposome size. Our fluorescence anisotropy assay can be used as a general method to investigate molecule-membrane interactions or molecule-molecule interactions in a high-throughput manner in nanometer-scaled containers like liposomes.

Direct observation of proton pumping by a eukaryotic P-type ATPase.

In eukaryotes, P-type adenosine triphosphatases (ATPases) generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. We monitored at the single-molecule level the activity of the prototypic proton-pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements, combined with a physical nonequilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 seconds) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters.

Single vesicle biochips for ultra-miniaturized nanoscale fluidics and single molecule bioscience.

One of the major bottlenecks in the development of biochips is maintaining the structure and function of biomolecules when interfacing them with hard matter (glass, plastics, metals, etc.), a challenge that is exacerbated during miniaturization that inevitably increases the interface to volume ratio of these devices. Biochips based on immobilized vesicles circumvent this problem by encapsulating biomolecules in the protective environment of a lipid bilayer, thus minimizing interactions with hard surfaces. Here we review the development of biochips based on arrays of single nanoscale vesicles, their fabrication via controlled self-assembly, and their characterization using fluorescence microscopy. We also highlight their applications in selected fields such as nanofluidics and single molecule bioscience. Despite their great potential for improved biocompatibility, extreme miniaturization and high throughput, single vesicle biochips are still a niche technology that has yet to establish its commercial relevance.