A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells

Hinton, Antentor; Katti, Prasanna; Christensen, Trace; Mungai, Margaret; Shao, Jianqiang; Zhang, Liang; Trushin, Sergey; Alghanem, Ahmad F.; Jaspersen, Adam; Geroux, Rachel E.; Neikirk, Kit; Biete, Michelle; Lopez, Edgar Garza; Shao, Bryanna; Vue, Zer; Vang, Larry; Beasley, Heather K.; Marshall, Andrea G.; Stephens, Dominique; Damo, Steven M.; Ponce, Jessica; Bleck, Christopher; Hicsasmaz, Innes; Murray, Sandra A.; Edmonds, Ranthony A. C.; Dajles, Andres; Koo, Young Do; Bacevac, Serif; Salisbury, Jeffrey L.; Pereira, Renata O.; Trushina, Eugenia; Abel, E. Dale

doi:10.1002/adbi.202200202

Cited by 25 publications

(23 citation statements)

References 89 publications

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“…Samples were processed as previously described by us (Pereira et al, 2017). Soleus samples were fixed with Trump's TEM fixative as previously described (Hinton et al, 2023) and stained. Random images of intermyofibrillar sections were obtained.…”

Section: Sample Preparation For Transmission Electron Microscope (Tem...mentioning

confidence: 99%

ATF4‐dependent increase in mitochondrial‐endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle

Hinton,

Katti,

Mungai

et al. 2024

Journal Cellular Physiology

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Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein‐ and lipid‐enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle‐specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin‐2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1‐loss‐induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4‐dependent manner.

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Section: Sample Preparation For Transmission Electron Microscope (Tem...mentioning

confidence: 99%

ATF4‐dependent increase in mitochondrial‐endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle

Hinton,

Katti,

Mungai

et al. 2024

Journal Cellular Physiology

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“…We measured 1250 mitochondria across 10 cells, with loss of Opa1 as a positive control for mitochondrial morphological changes because in vitro deletion of Opa1 alters mitochondrial morphology (Hinton et al, 2023;Lam et al, 2021;Pereira et al, 2017). Although…”

Section: D and 3d Structural Changes In Cristae And Mitochondria Afte...mentioning

confidence: 99%

“…SBF-SEM preparation was performed as described previously (Garza-Lopez et al, 2022;Hinton et al, 2023;Neikirk et al, 2021).…”

Section: Serial Block-face Scanning Electron Microscope (Sbf-sem) Pro...mentioning

confidence: 99%

3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex

Vue,

Garza‐Lopez,

Neikirk

et al. 2023

Aging Cell

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During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block‐face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase‐quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age‐related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age‐related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue‐dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

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Section: Sample Preparation For Transmission Electron Microscope (Tem...mentioning

confidence: 99%

ATF4 Dependent Increase in Mitochondrial-Endoplasmic Reticulum Tethering Following OPA1 Deletion in Skeletal Muscle

Hinton

Katti

Mungai

et al. 2022

Preprint

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Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate intracellular communications, contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and upregulated Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells induces MERC formation using multiple experimental models, including mice, Drosophila, and primary myotubes. Our results revealed that OPA1 deficiency increased MERC tethering and activated the integrated stress response (ISR) pathway effector, activating transcription factor 4 (ATF4). Loss of OPA1, reduced mitochondria calcium uptake and caffeine-induced calcium release by the ER, which was associated with changes in expression of mediators of calcium exchange IP3R3, GRP75, VDAC, at MERCs. Reducing Atf4 expression in Opa1-deficient muscle cells altered MERC ultrastructure changes and reestablished cytosolic calcium homeostasis. These data identify a role for ATF4 in the regulation of MERCs distance and mitochondrial calcium exchange.

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A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells

Cited by 25 publications

References 89 publications

ATF4‐dependent increase in mitochondrial‐endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle

ATF4‐dependent increase in mitochondrial‐endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle

3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex

ATF4 Dependent Increase in Mitochondrial-Endoplasmic Reticulum Tethering Following OPA1 Deletion in Skeletal Muscle

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