Direct Imaging of Liquid Domains in Membranes by Cryo Electron Tomography, bioRxiv, 2020-02-06

ABSTRACTImages of micron-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains’ shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been lacking, leading to an inability to answer key questions. For example, researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two new methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under native solution conditions. The first method is optimized for probe-free applications domains are directly distinguished from the surrounding membrane by their thickness. This technique measures area fractions of domains with quantitative accuracy, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to the first direct and quantitative imaging of submicron membrane domains under native conditions.SIGNIFICANCE STATEMENTFluorescence micrographs that capture the sizes, shapes, and distributions of liquid domains in model membranes have provided high standards of evidence to prove (and disprove) theories of how micron-scale domains form and grow. Corresponding theories about smaller domains have remained untested, partly because experimental methods of identifying submicron domains in vesicles under native solvent conditions have not been available. Here we introduce two such methods. Both leverage cryo-electron tomography to observe membrane features far smaller than the diffraction limit of light. The first method is probe-free and identifies differences in thicknesses between liquid domains and their surrounding membranes. The second method identifies membrane regions labeled by an electron-dense, fluorescent protein, which enables direct comparison of fluorescence micrographs with cryo-electron tomograms.

biorxiv biophysics 100-200-users 2020

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy, bioRxiv, 2020-02-06

ABSTRACTThe 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 Å, 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.

biorxiv biophysics 100-200-users 2020

 

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