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 cell types 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 generate MITO-Tag Mice. 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.

Mitochondrial Proteome of Affected Glutamatergic Neurons in a Mouse Model of Leigh Syndrome

“…Here, we have developed and implemented a novel viral vector approach (AAV-mitoTag) to purify cell type-specific mitochondria in a fast and minimally disruptive manner. Other genetic tools to isolate mitochondria with cell-type specificity have recently been developed (Bayraktar et al, 2019;Fecher et al, 2019). However, these rely on breeding strategies with mouse lines that have the potential disadvantage of introducing experimental confounds, such as germline recombination or transient expression of Cre recombinase during development, therefore compromising the cell type specificity of these approaches (Lam et al, 2011;Sanz et al, 2015).…”

A PEROXO-Tag enables rapid isolation of peroxisomes from human cells

“…Building upon our prior work utilizing epitope tags for rapid isolation of mitochondria (2,4) and lysosomes (5), we have now developed a PEROXO-Tag and PEROXO-IP workflow that allows for rapid (~10 min post-homogenization), specific, and facile isolation of peroxisomes from cultured human cells in a highly consistent manner.…”

“…We have previously developed multiple workflows that utilize 3XHA epitope tags localized to either mitochondria (2)(3)(4) or lysosomes (5) to enable rapid immunocapture of the respective subcellular compartment. These methods have been successfully used in conjunction with downstream metabolomic analyses to study the dynamics of mitochondria (2,4) and with metabolomic and proteomic analyses to study the behavior of lysosomes (5)(6)(7) and identify changes that were not readily visible with whole-cell or whole-tissue analyses. Historically, isolating peroxisomes has been one of the more challenging enterprises in organellar purification because peroxisomes can be fragile and share similar biophysical properties (e.g., density) with other organelles, such as mitochondria and lysosomes (8).…”

mSphere of Influence: Tweaking Organellar Purification Approaches

“…The group was able to rapidly isolate lysosomes (2) and peroxisomes (3). Former members of the Sabatini group also generated a MITO-tag mouse, allowing for the specific isolation of mouse hepatocyte mitochondria in vivo to assess their metabolome (4). As the techniques and workflows for organellar isolation continue to evolve, other approaches have been recently developed.…”