Skinkle, A. D. | bioRxiv Science

bioRxiv2020

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryoEM) can be used to directly image coexisting nanoscopic domains in synthetic and bio-derived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying lengths, we demonstrate that cryoEM can distinguish bilayer thickness differences as small as 0.5 [A], comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryoEM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, these methods reveal similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization. SIGNIFICANCEWe have used cryoEM to achieve direct, probe-free imaging of lateral domains in biomimetic lipid membranes under native conditions and to characterize differences in their structures. First, measurements of membrane thickness in laterally uniform single-component membranes show that cryoEM is capable of sub-angstrom resolution of interleaflet membrane thickness. All-atom simulations are used to predict the cryo-EM appearance of submicron domains in vesicles with coexisting liquid domains and these are quantitatively validated by direct imaging of phase separated membranes. We then extend this approach to observe nanoscopic domains in isolated cellular membranes, comprising the first direct imaging of nanodomains in biomembranes.

biophysics↗

bioRxiv2019

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Giant Plasma Membrane Vesicles (GPMVs) are a widely used model system for biochemical and biophysical analysis of the isolated mammalian plasma membrane (PM). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the plasma membrane while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes, and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed; i.e. that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic giant unilamellar vesicles (GUVs). We attribute this leakiness to relatively large and heterogeneous pores formed by rupture of vesicles from cells. These pores are stable and persist throughout experimentally relevant time scales. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration are of critical importance for interpreting experiments utilizing GPMVs as plasma membrane models, particularly for drug permeation and membrane asymmetry.\n\nSTATEMENT OF SIGNIFICANCEA critical assumption in using Giant Plasma Membrane Vesicles to study membrane penetration and interactions is that these vesicles maintain the permeability barrier of the native membrane from which they form. Using large fluorescently-labeled hydrophilic probes, we demonstrate that this assumption is often incorrect and conclude that macromolecular solutes permeate GPMVs through stable pores formed during shear-induced rupture of vesicles from cells. Using these insights into the mechanisms of poration, we demonstrate an approach to isolate sealed GPMVs.

biophysics↗