Serial cryoFIB/SEM Reveals Cytoarchitectural Disruptions in Leigh Syndrome Patient Cells

Zhu, Yanhua; Sun, Dapeng; Schertel, Andreas; Ning, Jiying; Fu, Xiaofeng; Gwo, Pam Pam; Watson, Alan M.; Zanetti-Domingues, Laura C.; Martin-Fernandez, Marisa L.; Freyberg, Zachary; Zhang, Peijun

doi:10.1016/j.str.2020.10.003

Cited by 33 publications

(20 citation statements)

References 29 publications

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“…As illustrated in the work ow (Figure S1), cryoEM grids containing SARS-CoV-2 infected cells were rst imaged in a Titan Krios to identify each individual infected cell (39.2 % for MOI of 0.1 and 45.4% for MOI 0.5) where cryoET tilt series were collected rst at the cell periphery. The grids were then transferred to a FIB/SEM dualbeam instrument and the exact same infected cells were imaged with serial cryoFIB/SEM volume imaging 36 or cryoFIB milling of cellular lamellae at the target region where additional cryoET tilt series were collected 37 . Alternatively, we imaged the same infected cells on cryoEM grids by soft X-ray cryo-tomography 38 .…”

Section: Sars-cov-2 Replication Induces Profound Cytopathic Effects Imentioning

confidence: 99%

Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress

Zhang

Mendonça

Howe

et al. 2021

Preprint

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Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events – e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus - in the context of whole-cell images. The latter revealed numerous heterogeneous cytoplasmic vesicles, the formation of membrane tunnels through which viruses exit, and the drastic cytoplasm invasion into the nucleus. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

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Section: Sars-cov-2 Replication Induces Profound Cytopathic Effects Imentioning

confidence: 99%

Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress

Zhang

Mendonça

Howe

et al. 2021

Preprint

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“…Cellular ultrastructure was visualized by detecting low energy secondary electrons without any additional treatment to enhance contrast ( Schertel et al., 2013 ). Subsequently, this method was applied to several biological systems ( Steyer et al., 2019 ; Spehner et al., 2020 ; Zhu et al., 2021 ). The large volume cellular context of vitrified biological specimens can be visualized using cryo-FIB-SEM volume imaging, with a slice thickness down to 10 nm and a lateral pixel size of >5 nm ( Vidavsky et al., 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans

Sexton

Burgold

Schertel

et al. 2022

Current Research in Structural Biology

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Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans . Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.

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“…Cryo-CLEM has proven to be a powerful method for in situ structural studies, including time-dependent events, especially those associated with viral entry, replication, and egress ( Briegel et al, 2010 ; Carter et al, 2020 ; Hampton et al, 2017 ; Jun et al, 2011 , 2019 ; Zhang, 2013 ). For samples that are too thick for direct cryo-EM imaging, cryo-sectioning ( Al-Amoudi et al, 2004 ; Bharat et al, 2018 ; Chlanda & Sachse, 2014 ; Salje et al, 2009 ; Zuber et al, 2008 ) and/or cryo-focused-ion-beam (FIB) milling ( Arnold et al, 2016 ; Gorelick et al, 2019 ; Hayles and De Winter, 2021 ; Hoffman et al, 2020 ; Hsieh et al, 2014 ; Mahamid et al, 2015 ; Marko et al, 2007 ; Rigort et al, 2012a , 2012b , 2010 ; Schaffer et al, 2019 ; Wagner et al, 2020 ; Wu et al, 2020 ; Zhu et al, 2021 ) may be used to expand the applicability of cryo-CLEM and cryo-ET.…”

Section: Introductionmentioning

confidence: 99%