Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism

Ahier, Arnaud; Dai, Chuan-Yang; Tweedie, Andrea; Bezawork-Geleta, Ayenachew; Kirmes, Ina; Zuryn, Steven

doi:10.1038/s41556-017-0023-x

Cited by 81 publications

(67 citation statements)

References 46 publications

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“…To perform more accurate purification of mitochondrial fraction to confirm mitochondrial localization of SESN2, we performed high affinity purification of mitochondria using magnetic beads bearing antibodies against mitochondrial outer membrane protein TOMM22. The effectiveness of this approach was verified in several recent studies and is considered to be of full value an alternative to ultracentrifuge method [27,28]. We observed that considerable amount of SESN2 was co-purified with the mitochondrial fraction supporting the previous observation that the protein is associated with mitochondrial fraction ( Fig 1B).…”

Section: Purification Of Sesn2 In Mitochondrial Fractionsupporting

confidence: 87%

Mitochondrial localization of SESN2

Kovaleva

Tokarchuk

Zeltuckin

et al. 2019

Preprint

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SESN2 is a member of evolutionarily conserved sestrin protein family found in most of Metazoa species. SESN2 is transcriptionally activated by many stress factors including metabolic derangements, oxidants and DNA-damage. As a result, SESN2 controls ROS accumulation, metabolism and cell viability. The best known function of SESN2 is the regulation of mechanistic target of rapamycin complex 1 kinase (mTORC1) that plays the central role in the stimulation of cell growth and suppression of autophagy. SESN2 inhibits mTORC1 activity through interaction with the GATOR2 protein complex that suppresses an inhibitory effect of GATOR2 on the GATOR1 protein complex. GATOR1 inhibits mTORC1 through its GAP activity toward the small GTPase RagA/B which in complex with RagC/D proteins stimulate mTORC1 translocation to the lysosomes where this kinase is activated by small GTPase Rheb. Despite the well-established role of SESN2 in mTORC1 inhibition, the other SESN2 activities are not well characterised. We recently showed that SESN2 can control mitochondrial function and cell death via mTORC1-independent mechanisms and these activities might be explained by direct effects of SESN2 on mitochondria. In this work we examined mitochondrial localization of SESN2 and demonstrated that SESN2 is located on mitochondria and can be directly involved in the regulation of mitochondrial functions.

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Section: Purification Of Sesn2 In Mitochondrial Fractionsupporting

confidence: 87%

Mitochondrial localization of SESN2

Kovaleva

Tokarchuk

Zeltuckin

et al. 2019

Preprint

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“…Methods for interrogating organellar metabolites with cell-type-specificity in vivo have thus far been limited in mammalian systems (9)(10)(11)(12)(13)(14). Building on our prior methodology using epitope-tags for fast isolations of mitochondria from cultured cells (2, 4), we have generated MITO-Tag Mice that allow for the rapid, cell-type-specific immunopurification and multimodal analysis of mammalian mitochondria in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…This would be useful for cell-types such as neurons, as the state of these cells can be particularly vulnerable to distortions during traditional cell-sorting methods due to their frailty and sprawling cellular architecture (8). To date however, no such methodology exists for rapidly isolating mitochondria with cell-type-specificity from mammalian tissues in vivo (9)(10)(11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Bayraktar

Baudrier

Özerdem

et al. 2018

Preprint

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Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific celltypes within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag ("MITO-Tag") for the fast isolation of mitochondria from cultured cells to now generate "MITO-TagMice." Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

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“…1E). This method is well established for mitochondria based on anti-Tom22 microbeads (29,30), but has recently been expanded to neoantigens (22,24). Indeed, for Emx1:Cre/GFP-OMM cortex, Western blot analysis corroborated efficient mitochondrial enrichment via IC GFP ( Fig.…”

Section: Mitotag Mice Allow Innocuously Tagging Of Cell Type-specificmentioning

confidence: 92%

“…This approach has been recently extended to organelles, e.g. to explore mitochondrial (22) and lysosomal (23) metabolomes in vitro, organellar DNA in invertebrates (24) or to correlate transcriptional and epigenetic signatures (25). To study mitochondrial diversity among cell types in situ, i.e.…”

Section: Introductionmentioning

confidence: 99%

Profound functional and molecular diversity of mitochondria revealed by cell type-specific profiling in vivo

Fecher

Trovò

Müller

et al. 2018

Preprint

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Mitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. Based on predictions from these proteomes, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons. Moreover, we identified Rmdn3 as a major determinant of ER-mitochondria proximity in Purkinje cells. Our novel approach enables exploring mitochondrial diversity on the functional and molecular level in many in vivo contexts.

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Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism

Cited by 81 publications

References 46 publications

Mitochondrial localization of SESN2

Mitochondrial localization of SESN2

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Profound functional and molecular diversity of mitochondria revealed by cell type-specific profiling in vivo

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