Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Visualizing membrane trafficking through the electron microscope: cryo-tomography of coat complexes.

ABSTRACT: Coat proteins mediate vesicular transport between intracellular compartments, which is essential for the distribution of molecules within the eukaryotic cell. The global arrangement of coat proteins on the membrane is key to their function, and cryo-electron tomography and subtomogram averaging have been used to study membrane-bound coat proteins, providing crucial structural insight. This review outlines a workflow for the structural elucidation of coat proteins, incorporating recent developments in the collection and processing of cryo-electron tomography data. Recent work on coat protein I, coat protein II and retromer performed on in vitro reconstitutions or in situ is summarized. These studies have answered long-standing questions regarding the mechanisms of membrane binding, polymerization and assembly regulation of coat proteins.

SUBMITTER: Markova EA

PROVIDER: S-EPMC6503763 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Visualizing membrane trafficking through the electron microscope: cryo-tomography of coat complexes.

Markova Evgenia A EA Zanetti Giulia G

Acta crystallographica. Section D, Structural biology 20190430 Pt 5

Coat proteins mediate vesicular transport between intracellular compartments, which is essential for the distribution of molecules within the eukaryotic cell. The global arrangement of coat proteins on the membrane is key to their function, and cryo-electron tomography and subtomogram averaging have been used to study membrane-bound coat proteins, providing crucial structural insight. This review outlines a workflow for the structural elucidation of coat proteins, incorporating recent developmen ...[more]

PMID: 31063149

Similar Datasets

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.

Project description:Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.

| S-EPMC6173284 | biostudies-literature

Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography.

Project description:The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

| S-EPMC10482868 | biostudies-literature

Visualizing insulin vesicle neighborhoods in β cells by cryo-electron tomography.

Project description:Subcellular neighborhoods, comprising specific ratios of organelles and proteins, serve a multitude of biological functions and are of particular importance in secretory cells. However, the role of subcellular neighborhoods in insulin vesicle maturation is poorly understood. Here, we present single-cell multiple distinct tomogram acquisitions of β cells for in situ visualization of distinct subcellular neighborhoods that are involved in the insulin vesicle secretory pathway. We propose that these neighborhoods play an essential role in the specific function of cellular material. In the regions where we observed insulin vesicles, a measurable increase in both the fraction of cellular volume occupied by vesicles and the average size (diameter) of the vesicles was apparent as sampling moved from the area near the nucleus toward the plasma membrane. These findings describe the important role of the nanometer-scale organization of subcellular neighborhoods on insulin vesicle maturation.

| S-EPMC7725471 | biostudies-literature

Visualizing active membrane protein complexes by electron cryotomography.

Project description:Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.

| S-EPMC4090714 | biostudies-literature

Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly.

Project description:Clathrin-coated vesicles mediate vesicular traffic in cells. Three-dimensional image reconstructions of homogenous populations of in vitro assembled clathrin coats have yielded a molecular model for clathrin and its interactions with some of its partners. The intrinsic averaging required for those calculations has precluded detailed analysis of heterogeneous populations of clathrin-coated vesicles isolated from cells. We have therefore used cryo-electron tomography to study the lattice organization of individual clathrin-coated vesicles and the disposition of the captured vesicle with respect to the surrounding coat. We find a wide range of designs for the clathrin lattice, with different patterns of pentagonal, hexagonal, and occasionally heptagonal facets. Many coats, even smaller ones, enclose membrane vesicles, which are generally offset from the center of the clathrin shell. The electron density distribution between the coat and the underlying vesicle is not uniform, and the number of apparent contacts that anchor the clathrin lattice to the vesicle membrane is significantly less than the number of clathrin heavy chains in the assembly. We suggest that the eccentric position of the vesicle reflects the polarity of assembly, from initiation of coat formation to membrane pinching.

| S-EPMC1839968 | biostudies-literature

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography.

Project description:The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

| S-EPMC8455137 | biostudies-literature

Reliable estimation of membrane curvature for cryo-electron tomography.

Project description:Curvature is a fundamental morphological descriptor of cellular membranes. Cryo-electron tomography (cryo-ET) is particularly well-suited to visualize and analyze membrane morphology in a close-to-native state and molecular resolution. However, current curvature estimation methods cannot be applied directly to membrane segmentations in cryo-ET, as these methods cannot cope with some of the artifacts introduced during image acquisition and membrane segmentation, such as quantization noise and open borders. Here, we developed and implemented a Python package for membrane curvature estimation from tomogram segmentations, which we named PyCurv. From a membrane segmentation, a signed surface (triangle mesh) is first extracted. The triangle mesh is then represented by a graph, which facilitates finding neighboring triangles and the calculation of geodesic distances necessary for local curvature estimation. PyCurv estimates curvature based on tensor voting. Beside curvatures, this algorithm also provides robust estimations of surface normals and principal directions. We tested PyCurv and three well-established methods on benchmark surfaces and biological data. This revealed the superior performance of PyCurv not only for cryo-ET, but also for data generated by other techniques such as light microscopy and magnetic resonance imaging. Altogether, PyCurv is a versatile open-source software to reliably estimate curvature of membranes and other surfaces in a wide variety of applications.

| S-EPMC7444595 | biostudies-literature

Cryo-electron tomography of the fungal membrane-assembled retromer:Grd19 coat containing Kex2 cargo motif

Project description: Not available

| EMPIAR-10631 | biostudies-other

Cryo-electron tomography of the metazoan membrane-assembled retromer:SNX3 coat containing Wls cargo motif

Project description: Not available

| EMPIAR-10633 | biostudies-other

Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography.

Project description:Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function.

| S-EPMC6252266 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data