Direct imaging of liquid domains in membranes by cryo-electron tomography

Abstract

Images of micrometer-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 limited. 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 methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, 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 direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.

Full scale structural, mechanical and dynamical properties of HIV-1 liposomes

“…The values were found to fluctuate within 34 to 36 Å. The observed overall thickness and subsequently small variations is in agreement with observations made by cryo-electron tomography for vesicles with a 42% molar ratio of cholesterol [33]. No relationship between local thickness values and composition was found in our analyses.…”

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

“…Consistent with its potential role as a cytoplasmic conduit into the EMC, the EMC3 portion of the cytoplasmic domain, which delineates this opening, sits approximately 45 Å from the lumenal side of the gated cavity. This dimension exceeds the thickness of the ER membrane ( Mitra et al, 2004 ; Heberle et al, 2020 ; Cornell et al, 2020 ; Figure 4D ). This cavity is lined primarily by EMC3, EMC4, and EMC6 ( Figure 5A ).…”

Regulation of Death Receptor Signaling by S-Palmitoylation and Detergent-Resistant Membrane Micro Domains—Greasing the Gears of Extrinsic Cell Death Induction, Survival, and Inflammation

“…This correlates with the thickness of the respective membranes, which are approximately 37.5 Å at the ER and 42.5 Å at the PM. Lipid rafts are supposed to be even thicker due to their high amount of sphingolipids and sterols, which can be monitored using, i.e., atomic force microscopy or cryo-electron tomography [ 17 , 18 , 19 , 20 ]. S-palmitoylation of proteins can for example induce tilting of TMDs having a positive HM to make them “shorter” or increase the hydrophobicity of proteins with a negative TM to allow residence in thick membranes (i.e., lipid rafts).…”