Staff member

Metabolism in biological systems involves the continuous formation and breakdown of chemical and structural components, driven by chemical energy. In specific, metabolic processes on cellular membranes result in in situ formation and degradation of the constituent phospholipid molecules, by consuming fuel, to dynamically regulate the properties. Synthetic analogs of such chemically fueled phospholipid vesicles have been challenging. Here we report a bio-inspired approach for the in situ formation of phospholipids, from water soluble precursors, and their fuel driven self-assembly into vesicles. We show that the kinetic competition between anabolic and catabolic-like reactions leads to the formation and enzymatic degradation of the double-tailed, vesicle-forming phospholipid. Spectroscopic and microscopic analysis demonstrate the formation of transient vesicles whose lifetime can be easily tuned from minutes to hours. Importantly, our design results in the formation of uniform sized (65 nm) vesicles simply by mixing the precursors, thus avoiding the traditional complex methods. Finally, our sub-100 nm vesicles are of the right size for application in drug delivery. We have demonstrated that the release kinetics of the incorporated cargo molecules can be dynamically regulated for potential applications in adaptive nanomedicine.

JTD Keywords: Droplets, Mode, Phospholipids, Supramolecular chemistry, Systems chemistry, Transient assembl, Vesicles

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ⁓1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

JTD Keywords: amplitude-modulation am-afm, atomic-force microscopy, capillary, force reconstruction, multifrequency afm, nanoscale wetting, persistence, reconstruction, relative-humidity, surfaces, tobacco mosaic virus (tmv), tobamovirus, transmission, water, Amplitude-modulation am-afm, Force reconstruction, Humidity, Microscopy, atomic force, Multifrequency afm, Nanoscale wetting, Tobacco mosaic virus, Tobacco mosaic virus (tmv), Tobacco mosaic virus (tmv), nanoscale wetting, Tobacco-mosaic-virus, Virion, Water, Wettability

This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ~100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors.

JTD

We measured and quantified the local electric polarization properties of ultrathin (~ 5 nm) biolayers on mm-thick mica substrates. We achieved it by scanning a sharp conductive tip (< 10 nm radius) of an electrostatic force microscope over the biolayers and quantifying sub-picoNewton electric polarization forces with a sharp-tip model implemented using finite-element numerical calculations. We obtained relative dielectric constants ?r = 3.3, 2.4 and 1.9 for bacteriorhodopsin, dioleoylphosphatidylcholine (DOPC) and cholesterol layers, chosen as representative of the main cell membrane components, with an error below 10% and a spatial resolution down to ~ 50 nm. The ability of using insulating substrates common in biophysics research, such as mica or glass, instead of metallic substrates, offers both a general platform to determine the dielectric properties of biolayers and a wider compatibility with other characterization techniques, such as optical microscopy. This opens up new possibilities for biolayer research at the nanoscale, including nanoscale label-free composition mapping.

JTD

Mechanical properties of nano-sized vesicles made up of natural membranes are crucial to the development of stable, biocompatible nanocontainers with enhanced functional, recognition and sensing capabilities. Here we measure and compare the mechanical properties of plasma and inner membrane nanovesicles ∼80 nm in diameter obtained from disrupted yeast Saccharomyces cerevisiae cells. We provide evidence of a highly deformable behaviour for these vesicles, able to support repeated wall-to-wall compressions without irreversible deformations, accompanied by a noticeably high Young's modulus (∼300 MPa) compared to that obtained for reconstituted artificial liposomes of similar size and approaching that of some virus particles. Surprisingly enough, the results are approximately similar for plasma and inner membrane nanovesicles, in spite of their different lipid compositions, especially on what concerns the ergosterol content. These results point towards an important structural role of membrane proteins in the mechanical response of natural membrane vesicles and open the perspective to their potential use as robust nanocontainers for bioapplications.

JTD

Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for sensing biodevices. This is particularly true for the case of G-protein coupled receptors (GPCRs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. However, the controlled production of natural vesicles containing GPCRs and their reproducible deposition on biosensor surfaces are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. In this work we present the production and characterization of membrane nanovesicles from Saccharomyces cerevisiae containing heterologously expressed olfactory receptors - a member of the family of GPCRs - and study their deposition onto substrates used as biosensor supports. We show by direct observation with Atomic Force Microscopy that nanovesicles deposit and flatten without rupturing on glass substrates following approximately a diffusive law. We show that surface coverages larger than 20-25% of the substrate can be reproducibly achieved under practical nanovesicle concentrations and reasonable time scales, while keeping to the minimum the presence of background residuals coming from the nanovesicles production process. Surface chemistry modification of gold substrates indicates a higher affinity of natural nanovesicles for acid modified surfaces as compared to amino or alcohol modified surfaces. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G-protein coupled membrane receptors.

JTD