MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection

Presley, Andrew D.; Fuller, Kathryn M.; Arriaga, Edgar A.

doi:10.1016/s1570-0232(03)00371-4

Cited by 157 publications

(138 citation statements)

References 37 publications

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“…We first examined the mitochondria in Atg7 f/f and Atg7 f/f Lck-Cre mature T cells using the relatively potential-independent mitochondria-specific vital dye MitoTracker Green (49). MitoTracker Green is a lipophilic thiol-reactive dye that selectively labels mitochondrial inner membrane and matrix (50) and has been used for measuring mitochondrial content in hematopoietic cells (51)(52)(53). Analysis by flow cytometry revealed a 50 -150% increase in the MitoTracker Green staining of Atg7-deficient CD4 ϩ and CD8 ϩ T lymphocytes in the spleen and lymph nodes (Fig.…”

Section: Enhanced Mitochondrial Content In Atg7-deficient Mature T Cellsmentioning

confidence: 99%

Autophagy Is Essential for Mitochondrial Clearance in Mature T Lymphocytes

Pua

Guo

Komatsu

et al. 2009

The Journal of Immunology

379

442

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Macroautophagy plays an important role in the regulation of cell survival, metabolism, and the lysosomal degradation of cytoplasmic material. In the immune system, autophagy contributes to the clearance of intracellular pathogens, MHCII cross-presentation of endogenous Ags, as well as cell survival. We and others have demonstrated that autophagy occurs in T lymphocytes and contributes to the regulation of their cellular function, including survival and proliferation. Here we show that the essential autophagy gene Atg7 is required in a cell-intrinsic manner for the survival of mature primary T lymphocytes. We also find that mitochondrial content is developmentally regulated in T but not in B cells, with exit from the thymus marking a transition from high mitochondrial content in thymocytes to lower mitochondrial content in mature T cells. Macroautophagy has been proposed to play an important role in the clearance of intracellular organelles, and autophagy-deficient mature T cells fail to reduce their mitochondrial content in vivo. Consistent with alterations in mitochondrial content, autophagy-deficient T cells have increased reactive oxygen species production as well as an imbalance in pro- and antiapoptotic protein expression. With much recent interest in the possibility of autophagy-dependent developmentally programmed clearance of organelles in lens epithelial cells and erythrocytes, our data demonstrate that autophagy may have a physiologically significant role in the clearance of superfluous mitochondria in T lymphocytes as part of normal T cell homeostasis.

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Section: Enhanced Mitochondrial Content In Atg7-deficient Mature T Cellsmentioning

confidence: 99%

Autophagy Is Essential for Mitochondrial Clearance in Mature T Lymphocytes

Pua

Guo

Komatsu

et al. 2009

The Journal of Immunology

379

442

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“…We therefore asked whether the different impact of Atg7 in the thymic development of iNKT versus conventional T cells correlated with a distinct regulation of mitochondrial content and autophagy during development of the two populations. Toward this aim we stained cells with the lipophilic thiol-reactive dye MitoTracker Green that selectively labels the mitochondrial inner membrane and matrix (25) and that has previously been used to measure mitochondrial content in hematopoietic cells (14). We observed that within the thymus mature CD4…”

Section: Mitochondrial Content and Autophagy Are Developmentallymentioning

confidence: 99%

Essential role for autophagy during invariant NKT cell development

Salio

Puleston

Mathan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Autophagy is an evolutionarily conserved cellular homeostatic pathway essential for development, immunity, and cell death. Although autophagy modulates MHC antigen presentation, it remains unclear whether autophagy defects impact on CD1d lipid loading and presentation to invariant natural killer T (iNKT) cells and on iNKT cell differentiation in the thymus. Furthermore, it remains unclear whether iNKT and conventional T cells have similar autophagy requirements for differentiation, survival, and/or activation. We report that, in mice with a conditional deletion of the essential autophagy gene Atg7 in the T-cell compartment (CD4 Cre-Atg7), thymic iNKT cell development-unlike conventional T-cell development-is blocked at an early stage and mature iNKT cells are absent in peripheral lymphoid organs. The defect is not due to altered loading of intracellular iNKT cell agonists; rather, it is T-cell-intrinsic, resulting in enhanced susceptibility of iNKT cells to apoptosis. We show that autophagy increases during iNKT cell thymic differentiation and that it developmentally regulates mitochondrial content through mitophagy in the thymus of mice and humans. Autophagy defects result in the intracellular accumulation of mitochondrial superoxide species and subsequent apoptotic cell death. Although autophagy-deficient conventional T cells develop normally, they show impaired peripheral survival, particularly memory CD8 + T cells. Because iNKT cells, unlike conventional T cells, differentiate into memory cells while in the thymus, our results highlight a unique autophagy-dependent metabolic regulation of adaptive and innate T cells, which is required for transition to a quiescent state after population expansion.utophagy is an evolutionarily conserved catabolic process that, by facilitating the breakdown and recycling of damaged organelles and long-lived proteins, is essential to maintaining cellular homeostasis (1). The autophagy pathway is highly regulated during development and also by environmental factors such as nutrient availability/starvation, hypoxia, and reactive oxygen species (ROS). The process is controlled by a number of autophagy-related genes (Atg) and starts with the formation of a double-membraned vesicle (the autophagosome) engulfing the cytoplasmic components to be delivered to the lysosome for degradation. Formation of the autophagosome requires the concerted action of two ubiquitin-like conjugation systems in which Atg12 is covalently linked to Atg5 and Atg8 is conjugated to phosphatidylethanolamine (2, 3). Atg7 is a necessary catalyst in both conjugation systems and is therefore essential for autophagy (3).Due to its important role in cellular homeostasis, dysregulation of autophagy has been implicated in several pathological processes, including neurodegeneration, autoimmunity, cancer, and infection (4). Furthermore, autophagy plays an important role during immune responses by regulating pathogen handling and antigen presentation (5, 6). It is well established that different populations of αβ T lymph...

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“…MitoTracker ® dyes carry a thiol-reactive chloromethyl group that covalently binds to proteins within the mitochondria, thereby allowing the dye to be better retained within mitochondria. 32 For this reason, these dyes can be used to track morphological changes as well as changes in mitochondrial numbers or mass after the induction or inhibition of mitophagy. It should be noted that the MitoTracker ® dyes vary in their propensity to accumulate and to be retained after mitochondrial depolarization.…”

Section: Monitoring Mitophagy Through Colocalization Of Mitochondrialmentioning

confidence: 99%