Quantitative Cryo-Electron Tomography

Navarro, Paula P.

doi:10.3389/fmolb.2022.934465

Cited by 13 publications

(7 citation statements)

References 123 publications

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“…It is important to note that cryo-electron tomograms generated by cryo-FIB milling cover a section of the mitochondria contained within the thickness of the lamella, thus, all mitochondria were partially imaged in the Z-axis. In some cases, mitochondria are only partially visible in the XY plane due to the trade-off between resolution and field of view (Navarro, 2022 ; Appendix Fig. S2A ).…”

Section: Resultsmentioning

confidence: 99%

In situ architecture of Opa1-dependent mitochondrial cristae remodeling

Fry,

Navarro,

Hakim

et al. 2024

EMBO J

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Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

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Section: Resultsmentioning

confidence: 99%

In situ architecture of Opa1-dependent mitochondrial cristae remodeling

Fry,

Navarro,

Hakim

et al. 2024

EMBO J

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“…In some cases, mitochondria are only partially visible in the XY plane due to the trade-off between resolution and field of view (Navarro, 2022) (Fig. S2a).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

In situarchitecture of Opa1-dependent mitochondrial cristae remodeling

Fry

Navarro

Qin

et al. 2023

Preprint

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Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to the lack of native 3D views of cristae morphology. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe elongated mitochondria with a stacking phenotype, and an absence of tubular cristae, when only l-Opa1 is present. In contrast, when mitochondria contain mainly s-Opa1, we observe irregular cristae packing, an increase in globular cristae, and decreased matrix condensation. Notably, we find the absence of l-Opa1 results in mitochondria with wider cristae junctions. BH3 profiling reveals that absence of l-Opa1 reduces cytochrome c release in response to pro-apoptotic stimuli. We discuss the implications Opa1-dependent cristae morphologies in cell death initiation.

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“…Localizing regions of interest to mill lamellae has proven to be another significant challenge, particularly for multicellular and tissue specimens ( Navarro, 2022 ). While correlative techniques can aid in such a task, particularly developments incorporating super-resolution techniques, the limited number of fluorescent probes amenable to imaging at cryogenic temperatures limits the wider applicability of these techniques ( Dahlberg et al, 2018 ).…”

Section: Challenges and Limitations In Traditional And Cryogenic Micr...mentioning

confidence: 99%

The advent of preventive high-resolution structural histopathology by artificial-intelligence-powered cryogenic electron tomography

Galaz-Montoya

2024

Front. Mol. Biosci.

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Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by “big data” in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

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Quantitative Cryo-Electron Tomography

Cited by 13 publications

References 123 publications

In situ architecture of Opa1-dependent mitochondrial cristae remodeling

In situ architecture of Opa1-dependent mitochondrial cristae remodeling

In situarchitecture of Opa1-dependent mitochondrial cristae remodeling

The advent of preventive high-resolution structural histopathology by artificial-intelligence-powered cryogenic electron tomography

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