Estimation of Membrane Curvature for Cryo-Electron Tomography

AbstractCurvature is an important 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 high resolution. However, current curvature estimation methods cannot be applied directly to membrane segmentations in cryo-ET. Additionally, a reliable estimation requires to cope with quantization noise. Here, we developed and implemented a method for membrane curvature estimation from tomogram segmentations.From a membrane segmentation, a signed surface (triangle mesh) is first extracted. The triangle mesh is then represented by a graph (vertices and edges), which facilitates finding neighboring triangles and the calculation of geodesic distances necessary for local curvature estimation. Here, we present several approaches for accurate curvature estimation based on tensor voting. Beside curvatures, these methods also provide robust estimations of surface normals and principal directions.We tested the different methods on benchmark surfaces with known curvature, demonstrating the validity of these methods and their robustness to quantization noise. We also applied two of these approaches to biological cryo-ET data. The results allowed us to determine the best approach to estimate membrane curvature in cellular cryo-ET data.

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

Nature Communications ◽

10.1038/s41467-020-19619-7 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 4

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.

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Reliable estimation of membrane curvature for cryo-electron tomography

PLoS Computational Biology ◽

10.1371/journal.pcbi.1007962 ◽

2020 ◽

Vol 16 (8) ◽

pp. e1007962

Author(s):

Maria Salfer ◽

Javier F. Collado ◽

Wolfgang Baumeister ◽

Rubén Fernández-Busnadiego ◽

Antonio Martínez-Sánchez

Keyword(s):

Electron Tomography ◽

Membrane Curvature ◽

Reliable Estimation ◽

Cryo Electron Tomography

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Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

Journal of Virology ◽

10.1128/jvi.00947-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 23

Author(s):

Petr Chlanda ◽

Elena Mekhedov ◽

Hang Waters ◽

Alexander Sodt ◽

Cindi Schwartz ◽

...

Keyword(s):

Membrane Fusion ◽

Influenza A Virus ◽

Influenza A ◽

Electron Tomography ◽

Virus Assembly ◽

Membrane Curvature ◽

Fusion Pore ◽

M1 Protein ◽

Cryo Electron Tomography ◽

Pore Expansion

ABSTRACT The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.

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

10.1101/2020.06.23.167064 ◽

2020 ◽

Cited By ~ 25

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.

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Tricalbin-mediated contact sites control ER curvature to maintain plasma membrane integrity

10.1101/578427 ◽

2019 ◽

Author(s):

Javier Collado ◽

Maria Kalemanov ◽

Antonio Martínez-Sánchez ◽

Felix Campelo ◽

Wolfgang Baumeister ◽

...

Keyword(s):

Plasma Membrane ◽

Electron Tomography ◽

Membrane Integrity ◽

Membrane Curvature ◽

Quantitative Modeling ◽

Membrane Contact Sites ◽

Contact Sites ◽

Cryo Electron Tomography ◽

Membrane Contact ◽

Mediated Contact

SummaryMembrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. ER-PM MCS are particularly abundant in S. cerevisiae, where approximately half of the PM surface is covered by cortical ER (cER). Several proteins, including Ist2, Scs2/22 and Tcb1/2/3 are implicated in cER formation, but the specific roles of these molecules are poorly understood. Here we use cryo-electron tomography to show that ER-PM tethers are key determinants of cER morphology. In particular, Tcb proteins form peaks of extreme curvature on the cER membrane facing the PM. Semi-quantitative modeling and functional assays suggest that Tcb-mediated cER peaks facilitate the transport of lipids from the cER to the PM, necessary to maintain PM integrity under stress conditions. ER peaks were also present at other MCS, implying that membrane curvature enforcement may be a widespread mechanism to expedite lipid transport at MCS.

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Mechanistic insight into bacterial entrapment by septin cage reconstitution

Nature Communications ◽

10.1038/s41467-021-24721-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Damián Lobato-Márquez ◽

Jingwei Xu ◽

Gizem Özbaykal Güler ◽

Adaobi Ojiakor ◽

Martin Pilhofer ◽

...

Keyword(s):

Electron Tomography ◽

Shigella Flexneri ◽

Membrane Curvature ◽

Cytoskeletal Proteins ◽

Bacterial Cells ◽

Cryo Electron Tomography ◽

Curved Membranes ◽

Positively Curved ◽

Insight Into

AbstractSeptins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. We show that septin complexes recognize the pole of growing Shigella cells. An amphipathic helix domain in human SEPT6 enables septins to sense positively curved membranes and entrap bacterial cells. Shigella strains lacking lipopolysaccharide components are more efficiently entrapped in septin cages. Finally, cryo-electron tomography of in vitro cages reveals how septins assemble as filaments on the bacterial cell surface.

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