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

Abstract

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.

Neuronal metabolic rewiring promotes resilience to neurodegeneration caused by mitochondrial dysfunction

“…While absolute lack of contamination from other cell types is virtually impossible to achieve, especially from adult tissue, we combined an effective dissociation step with FACS to obtain sufficient amounts of viable neurons for downstream proteomic analysis with a reasonably high protein coverage (~3000 proteins) for a single cell population, in comparison with existing datasets from whole cerebellum (51). By preserving whole-cell viability, the approach we provide here allows us to examine not only changes in metabolic pathways within mitochondria but also their cytosolic counterparts, thus complementing well with recent methods that take advantage of mitochondrial membrane tags to enrich for cell type-specific populations of mitochondria from complex tissue (52,53). The method we describe not only will be relevant for studies of Purkinje cells but also can be easily adapted and used for any cell type to address metabolic alterations in the diseased brain, including in other models of mitochondrial dysfunction.…”

Proanthocyanidin Biosynthesis—a Matter of Protection

“…With a suitable model selected, the key approach to solving the problem of coupled PA synthesis and intracellular trafficking will be the proteomic and metabolomic characterization of the vesicles seen in the tannosome model and in the various mutants described above. Methods are available for labeling cellular organelles/compartments to allow for visualization of colocalization within the cell (Geldner et al, 2009) and affinity purification for biochemical analysis (Bayraktar et al, 2019;Xiong et al, 2019), and the sensitivity of both proteomics and metabolomics has now improved to the point where analysis of plant subcellular compartments is possible (Fürtauer et al, 2019). The vesicles purported to contain PAs in the tannosome model sedimented to the bottom of the ultracentrifuge tube during purification (Brillouet et al, 2014) and could therefore be contaminated with precipitated PAs.…”

Spatiotemporal regulation of NADP(H) phosphatase Nocturnin and its role in oxidative stress response

“…Even though the total NAD(H) pool in the mitochondria is much larger than NADP(H) due to the huge abundance of NAD + , NADPH and NADH levels in the mitochondria have been shown to be approximately equimolar (34). It is possible that NADPH de-phosphorylation is a mechanism for generating antiphasic rhythms in mitochondrial NADPH and NADH.…”

“…Bioinformatic analysis has predicted the existence of more than 300 NAD(H)-or NADP(H)-requiring enzymes, some of which are extremely abundant, and any of which can influence the resulting concentration measurements (16). Therefore, future studies that can employ methods of rapid mitochondrial isolation throughout the circadian cycle, such as the MitoTag system (34,35), will be of great importance in understanding the communication between circadian rhythms and mitochondrial metabolism.…”