scholarly journals Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network

2020 ◽  
Author(s):  
Joshua Hutchings ◽  
Viktoriya G. Stancheva ◽  
Nick R. Brown ◽  
Alan C.M. Cheung ◽  
Elizabeth A. Miller ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Interaction Network ◽  
Morphological Plasticity ◽  
Membrane Curvature ◽  
Coat Proteins ◽  
Physiological Importance ◽  
Structural Framework ◽  
Multiple Interfaces ◽  
Cargo Proteins ◽  
Subtomogram Averaging

AbstractThe COPII coat mediates Endoplasmic Reticulum (ER) to Golgi trafficking for thousands of proteins. Five essential coat proteins assemble at the ER into a characteristic two-layer architecture, which recruits cargo proteins whilst sculpting membrane carriers with diverse morphologies. How coat architecture drives membrane curvature whilst ensuring morphological plasticity is largely unknown, yet is central to understanding mechanisms of carrier formation. Here, we use an established reconstitution system to visualise the complete, membrane-assembled COPII coat with unprecedented detail by cryo-electron tomography and subtomogram averaging. We discover a network of interactions within and between coat layers, including multiple interfaces that were previously unknown. We reveal the physiological importance of these interactions using genetic and biochemical approaches. A newly resolved Sec31 C-terminal domain provides order to the coat and is essential to drive membrane curvature in cells. Moreover, a novel outer coat assembly mode provides a basis for coat adaptability to varying membrane curvatures. Furthermore, a newly resolved region of Sec23, which we term the L-loop, imparts coat stability and in part dictates membrane shape. Our results suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.

scholarly journals Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joshua Hutchings ◽  
Viktoriya G. Stancheva ◽  
Nick R. Brown ◽  
Alan C. M. Cheung ◽  
Elizabeth A. Miller ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Interaction Network ◽  
Membrane Curvature ◽  
Outer Coat ◽  
Functional Roles ◽  
Physiological Importance ◽  
Structural Framework ◽  
Subtomogram Averaging ◽  
Current Paradigm ◽  
Full Network

AbstractCOPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.

Assembly of COPI and COPII Vesicular Coat Proteins on Membranes

2018 ◽  
Vol 47 (1) ◽  
pp. 63-83 ◽  
Author(s):  
Julien Béthune ◽  
Felix T. Wieland
Keyword(s):  
Electron Tomography ◽  
Coat Proteins ◽  
Structural Elements ◽  
X Ray Crystallography ◽  
Transport Vesicles ◽  
Cryo Electron Tomography ◽  
Functional Implications ◽  
Cargo Proteins ◽  
Intracellular Compartments ◽  
Proteins And Peptides

In eukaryotes, distinct transport vesicles functionally connect various intracellular compartments. These carriers mediate transport of membranes for the biogenesis and maintenance of organelles, secretion of cargo proteins and peptides, and uptake of cargo into the cell. Transport vesicles have distinct protein coats that assemble on a donor membrane where they can select cargo and curve the membrane to form a bud. A multitude of structural elements of coat proteins have been solved by X-ray crystallography. More recently, the architectures of the COPI and COPII coats were elucidated in context with their membrane by cryo-electron tomography. Here, we describe insights gained from the structures of these two coat lattices and discuss the resulting functional implications.

scholarly journals SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steffen Klein ◽  
Mirko Cortese ◽  
Sophie L. Winter ◽  
Moritz Wachsmuth-Melm ◽  
Christopher J. Neufeldt ◽  
...  
Keyword(s):  
Viral Replication ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Membrane Curvature ◽  
Virion Assembly ◽  
Ribonucleoprotein Complexes ◽  
Cryo Electron Tomography ◽  
Membrane Bending ◽  
Subtomogram Averaging

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

scholarly journals SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

2020 ◽  
Author(s):  
Steffen Klein ◽  
Mirko Cortese ◽  
Sophie L. Winter ◽  
Moritz Wachsmuth-Melm ◽  
Christopher J. Neufeldt ◽  
...  
Keyword(s):  
Viral Replication ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Membrane Curvature ◽  
Virion Assembly ◽  
Ribonucleoprotein Complexes ◽  
Cryo Electron Tomography ◽  
Membrane Bending ◽  
Subtomogram Averaging

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualized RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We found that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

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

2019 ◽  
Vol 75 (5) ◽  
pp. 467-474 ◽  
Author(s):  
Evgenia A. Markova ◽  
Giulia Zanetti
Keyword(s):  
Coat Protein ◽  
Membrane Trafficking ◽  
Electron Tomography ◽  
Eukaryotic Cell ◽  
Coat Proteins ◽  
Cryo Electron Tomography ◽  
Recent Developments ◽  
Intracellular Compartments ◽  
Subtomogram Averaging

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.

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals Nuclear pores constrict upon energy depletion

2020 ◽  
Author(s):  
Christian E Zimmerli ◽  
Matteo Allegretti ◽  
Vasileios Rantos ◽  
Sara K Goetz ◽  
Agnieszka Obarska-Kosinska ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Electron Tomography ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Energy Status ◽  
Energy Depletion ◽  
Time Variations ◽  
Subtomogram Averaging ◽  
Pore Complexes

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins that form an intricate cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter were reported, but the physiological circumstances and the molecular details remain unknown. Here we combined cryo-electron tomography and subtomogram averaging with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. While actively transporting NPCs adopt a dilated conformation, they strongly constrict upon cellular energy depletion. Fluorescence recovery after photo bleaching experiments show that NPC constriction is concomitant with reduced diffusion and active transport across the nuclear envelope. Our data point to a model where the energy status of cells is linked to the conformation of NPC architecture.

scholarly journals A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

2015 ◽  
Vol 112 (12) ◽  
pp. E1443-E1452 ◽  
Author(s):  
Zhiyong Bai ◽  
Barth D. Grant
Keyword(s):  
Functional Module ◽  
Small Gtpase ◽  
Recycling Endosome ◽  
Vesicle Budding ◽  
Recycling Endosomes ◽  
Physiological Importance ◽  
Golgi Transport ◽  
Early Endosomes ◽  
Cargo Proteins ◽  
Signaling Receptors

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.

scholarly journals The Dynamo Software Package for Cryo-electron Tomography and Subtomogram Averaging

2020 ◽  
Vol 26 (S2) ◽  
pp. 3142-3145
Author(s):  
Paula Navarro ◽  
Stefano Scaramuzza ◽  
Henning Stahlberg ◽  
Daniel Castaño-Díez
Keyword(s):  
Software Package ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

scholarly journals A novel constrained reconstruction model towards high-resolution subtomogram averaging

2019 ◽  
Author(s):  
Renmin Han ◽  
Lun Li ◽  
Peng Yang ◽  
Fa Zhang ◽  
Xin Gao
Keyword(s):  
Averaging Method ◽  
Electron Tomography ◽  
Solution Space ◽  
Supplementary Information ◽  
Reconstruction Technique ◽  
Constrained Reconstruction ◽  
Kaczmarz Algorithm ◽  
Reconstruction Quality ◽  
Subtomogram Averaging ◽  
Reconstruction Model

Abstract Motivation Electron tomography (ET) offers a unique capacity to image biological structures in situ. However, the resolution of ET reconstructed tomograms is not comparable to that of the single-particle cryo-EM. If many copies of the object of interest are present in the tomograms, their structures can be reconstructed in the tomogram, picked, aligned and averaged to increase the signal-to-noise ratio and improve the resolution, which is known as the subtomogram averaging. To date, the resolution improvement of the subtomogram averaging is still limited because each reconstructed subtomogram is of low reconstruction quality due to the missing wedge issue. Results In this article, we propose a novel computational model, the constrained reconstruction model (CRM), to better recover the information from the multiple subtomograms and compensate for the missing wedge issue in each of them. CRM is supposed to produce a refined reconstruction in the final turn of subtomogram averaging after alignment, instead of directly taking the average. We first formulate the averaging method and our CRM as linear systems, and prove that the solution space of CRM is no larger, and in practice much smaller, than that of the averaging method. We then propose a sparse Kaczmarz algorithm to solve the formulated CRM, and further extend the solution to the simultaneous algebraic reconstruction technique (SART). Experimental results demonstrate that CRM can significantly alleviate the missing wedge issue and improve the final reconstruction quality. In addition, our model is robust to the number of images in each tilt series, the tilt range and the noise level. Availability and implementation The codes of CRM-SIRT and CRM-SART are available at https://github.com/icthrm/CRM. Contact [email protected] or [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

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