A comprehensive approach to artifact-free sample preparation and the assessment of mitochondrial morphology in tissue and cultured cells

Hinton, Antentor; Katti, Prasanna; Ta, Christensen; Mungai, Margaret; Shao, Jianqiang; Zhang, L; Trushin, Sergey; Alghanem, Ahmad F.; Jaspersen, Adam; Re, Geroux; Neikirk, Kit; Biete, Michelle; E, Garza Lopez; Z, Vue; Beasley, Heather K.; Marshall, Andrea G.; Hicsasmaz, Innes; Ra, Edmonds; Dajles, Andres; Yd, Koo; Bacevac, Serif; Jl, Salisbury; Ro, Pereira; Trushina, Eugenia

doi:10.1101/2021.06.27.450055

Cited by 11 publications

(30 citation statements)

References 73 publications

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“…Using CRISPR/Cas9, guide RNA (Table 2), and a control plasmid, we transiently knockout the genes for MICOS complex components and Opa-1 from skeletal muscle cells. As observed previously, in vitro deletion of OPA-1 altered mitochondrial morphology 16,28,29 .…”

Section: Resultssupporting

confidence: 85%

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3D Reconstruction of Murine Mitochondria Exhibits Changes in Structure Across Aging Linked to the MICOS Complex

Vue

Lopez

Neikirk

et al. 2022

Preprint

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Background: Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. Studies of bioenergetics and protein turnover show that mitochondria mediate this decline in function. Mitochondria are essential for the production of ATP, which occurs in the cristae, the folds of the inner mitochondrial membrane. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging are unknown. Further, we do not understand how 3D mitochondrial networks and the specialization of mitochondria alter during aging. Methods: We measure changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. Nanotunnels are also measured through 3D reconstruction. CRISPR/Cas9 KD is performed to examine changes in mitochondria upon loss of MICOS complex and OPA-1. Metabolomics are used to find key metabolite pathways changed upon MICOS complex loss. Results: We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, and cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle across aging. Cardiac muscle showed similar differences but less fragmentation and wide-spread changes across 2-year aaging. We also found an association of optic atrophy 1 (OPA-1) and the MICOS complex in the gastrocnemius with mitochondrial aging, decreased oxidative capacity, and altered mitochondrial metabolism. Conclusions: We are the first to examine mitochondria changes in skeletal muscle and cardiac muscle across aging. 3D reconstructions of nanotunnels elucidated novel patterns in skeletal muscle. Notably, we noticed differences in skeletal and cardiac muscle that suggests a differential response to mitochondrial aging in cardiac and skeletal muscle. Importantly, we found similar changes in mitochondrial morphology were observed in aging skeletal muscles and for loss of MICOS proteins in mouse skeletal muscle. Furthermore, MICOS proteins decreased with age. In tandem, this suggests a relationship between the MICOS complex and aging, which further 3D reconstruction could potentially further link to disease states.

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Section: Resultssupporting

confidence: 85%

“…Quantification of TEM images was performed as described previously using the National Institutes of Health (NIH) ImageJ software 16,28 . Cells were divided into four quadrants and two quadrants were selected randomly for complete analysis.…”

Section: Quantification Of Tem Micrographs and Parameters Using Imagejmentioning

confidence: 99%

3D Reconstruction of Murine Mitochondria Exhibits Changes in Structure Across Aging Linked to the MICOS Complex

Vue

Lopez

Neikirk

et al. 2022

Preprint

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“…Therefore, also using SBF-SEM for 3D reconstruction can allow for a more complete understanding of the mitochondria morphology. Future studies may consider also utilizing focused ion beam-scanning electron microscopy, which allows for the 3D reconstruction of smaller objects such as cristae 44 . In the past, FIB-SEM has successfully been performed in mouse muscle to characterize mitochondria 45 .…”

Section: Discussionmentioning

confidence: 99%

“…SBF-SEM was performed as described previously 16,44,46 . 5% isoflurane was used to anesthetize male mice.…”

Section: Methodsmentioning

confidence: 99%

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Three-Dimensional Mitochondria Reconstructions of Murine Cardiac Muscle Changes in Size Across Aging

Vue

Garza-López

Neikirk

et al. 2022

Preprint

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Cardiac disease remains a large cause of death among humans and therapies to treat it remain lacking. Mitochondria is heavily implicated in various critical functions of the cell not limited to the production of ATP. Critically, mitochondria break down across aging and heart also decrease in efficiency in aging. Key factors implicated in mitochondria morphology, such as the mitochondrial contact site and cristae organizing system (MICOS), role across aging remains to be seen in cardiac muscle. To better understand the relationship between mitochondria in heart, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the 3D networks in cardiac muscle samples of mice at aging intervals. Cardiac muscle showed some breaking down across aging, decreased areas, increased mitochondria, but less fragmentation and wide-spread changes across 2-year aging than previous studies in skeletal muscle. In studying cristae, the inner folds of mitochondria, we observed a loss of morphology across aging. This mimicked what was observed upon CRISPR/Cas9 knockdown of mitofilin, Chchd3, Chchd6 and Opa-1 which showed increased lamellar cristae and fragmented mitochondria while mitochondria length and volume decreased. We are the first to examine mitochondria changes in cardiac muscle across aging. Notably, we noticed differences in skeletal muscle, which we have previously studied, and cardiac muscle that suggests a differential response to mitochondrial aging in cardiac and skeletal muscle. In combination, these data suggest that, in the heart, loss of the MICOS complex may be implicated in the loss of function in mitochondria that is seen across aging.

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New Highly Sensitive and Specific Raman Probe for Live Cell Imaging of Mitochondrial Function

Pieczara,

Arellano Reyes,

Keyes

et al. 2024

ACS Sens.

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For Raman hyperspectral detection and imaging in live cells, it is very desirable to create novel probes with strong and unique Raman vibrations in the biological silent region (1800–2800 cm–1). The use of molecular probes in Raman imaging is a relatively new technique in subcellular research; however, it is developing very rapidly. Compared with the label-free method, it allows for a more sensitive and selective visualization of organelles within a single cell. Biological systems are incredibly complex and heterogeneous. Directly visualizing biological structures and activities at the cellular and subcellular levels remains by far one of the most intuitive and powerful ways to study biological problems. Each organelle plays a specific and essential role in cellular processes, but importantly for cells to survive, mitochondrial function must be reliable. Motivated by earlier attempts and successes of biorthogonal chemical imaging, we develop a tool supporting Raman imaging of cells to track biochemical changes associated with mitochondrial function at the cellular level in an in vitro model. In this work, we present a newly synthesized highly sensitive RAR-BR Raman probe for the selective imaging of mitochondria in live endothelial cells.

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A comprehensive approach to artifact-free sample preparation and the assessment of mitochondrial morphology in tissue and cultured cells

Cited by 11 publications

References 73 publications

3D Reconstruction of Murine Mitochondria Exhibits Changes in Structure Across Aging Linked to the MICOS Complex

3D Reconstruction of Murine Mitochondria Exhibits Changes in Structure Across Aging Linked to the MICOS Complex

Three-Dimensional Mitochondria Reconstructions of Murine Cardiac Muscle Changes in Size Across Aging

New Highly Sensitive and Specific Raman Probe for Live Cell Imaging of Mitochondrial Function

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