Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy
Summary
The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.
Abstract
Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid's position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.
Introduction
Circular 16.5 kbp DNA molecules constitute the genetic material of mitochondria, encoding 22 tRNAs, 2 rRNAs, and 13 polypeptides needed for mitochondrial oxidative phosphorylation complexes. Mitochondrial DNA bound to mitochondrial transcription factor a (TFAM) and several other proteins form the mitochondrial nucleoids1,2,3,4. Mitochondrial nucleoids moveand redistribute between the components of the mitochondrial network5,6 during its morphological remodeling, fission or fusion depending on cell cycle phase, stress, and other factors (reviewed in Pernas et al.7). In addition, the motion of mitochondrial nucleoids, is implicated in systemic lupus erythematosus disease8 and may play a role in other diseases. Fluorescence microscopy is a straightforward technique for live-cell studies of organelles, but the technique has a resolution of >200 nm, which is larger than the size of mitochondrial nucleoids (~100 nm9,10,11,12). This limit has been circumvented by so called "super-resolution" techniques, such as stimulated emission depletion (STED) and single molecule localization microscopy (SMLM)13,14. So far, mitochondrial nucleoids and other DNAs were imaged in live cells by direct stochastic optical reconstruction microscopy (dSTORM)15. Fine sub-mitochondrial structures with positions correlating with mtDNA were observed by STED in live cells16. However, these super-resolution techniques require high illumination intensity, which causes phototoxic effects on living cells17. Therefore, time lapse imaging of mitochondrial nucleoids with resolution beyond diffraction limit is challenging. To address this, we used super-resolution structured illumination microscopy (SR-SIM)18. SIM requires a much lower illumination power dose than STED and SMLM19. Furthermore, in contrast to STED and SMLM techniques, SIM permits straightforward multicolor three-dimensional (3D) imaging, and it does not require particular photophysical properties of the fluorophores or imaging buffer composition19.
The conventional strategy for labeling mitochondrial nucleoids in live cells is fluorescent tagging of a mitochondrial nucleoid protein, such as TFAM20. However, in many cases, this strategy is not suitable. Moreover, overexpression of fluorescent protein-tagged TFAM produces a serious artifact21. Labeling of DNA with organic dyes has advantages over a fluorescent protein (FP)-based strategy. Organic dyes are free of constrains related to FP tagging: they can be used for any type of cells or tissueand can beapplied at any time point of an experiment. Live cell imaging of mitochondrial nucleoids has been reported with several DNA-binding dyes: DAPI22, SYBR Green23, Vybrant DyeCycle24, and picoGreen15,25,26. A substantial drawback of most DNA-binding dyes for nucleoid labeling is that they stain all DNA within the cell. Targeting a dye solely to mitochondrial DNA is highly desirable. To achieve that, careful selection of a dye possessing suitable physico-chemical properties is necessary. Lipophilic dyes possessing delocalized positive charge, such as rhodamine 123, are known to accumulate in live mitochondria, which preserve their negative membrane potential. In addition, an ideal dye for specific labeling of mitochondrial nucleoids should bind DNA with high affinity and emit bright fluorescence upon DNA binding. Considering these requirements, certain cyanines are promising (e.g., picoGreen), but nuclear DNA is abundantly stained by these dyes simultaneously with mitochondrial DNA15,25,26. The present protocol describes specific labeling of mitochondrial nucleoids in live cells with another cyanine dye, SYBR Gold (SG), and tracking of the nucleoids in time lapse super-resolution SIM videos. Moreover, SG-stained live cells can be imaged by any type of inverted fluorescent microscope (confocal, spinning disk, epifluorescence, etc.) suitable for living cells and equipped with a 488 nm light source.
Protocol
Representative Results
Discussion
There are several critical components to the protocol: To achieve preferential labeling of mitochondrial DNA, the concentration of the DNA binding dye during incubation should be kept very low (e.g., a 1:10,000 dilution of a typical commercial stock), and the incubation time should be 30 min. The incubation time should never exceed 1 h. SYBR Gold dye should be used; other DNA-binding dyes are not bright enough to generate a strong signal upon labeling at a low concentration.
The limitation of …
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge Asifa Akhtar and Angelika Rambold (both Max Planck Institute for Immunobiology and Epigenetics) for providing HeLa cells.
Materials
| Elyra PS1 | Carl Zeiss | multi-modal super-resolution microscope containing module for super-resolution structured illumination microscopy (SR-SIM) | |
| high glucose DMEM | GIBCO/ThermoFisher | 31966021 | |
| ibidi 35-mm dish, glass bottom | Ibidi Gmbh | 81158 | |
| ibidi 8-well microSlide, glass bottom | Ibidi Gmbh | 80827 | |
| Imaris 8.4.1 | Bitplane/Oxford Instruments | image porcessing and visualisation software package | |
| iXon 885 | Andor Technologies | EMCCD camera with back-illuminated sensor | |
| LSM880 Airyscan | Carl Zeiss | laser scanning confocal microscope with array detector | |
| Mitotracker CMXRos Red | ThermoFischer | M7512 | red live cell mitochondrial stain |
| Mitotracker Deep Red FM | ThermoFischer | M22426 | far red live cell mitochondrial stain |
| picoGreen | ThermoFischer | P7581 | cell permeant DNA stain |
| Plan Apochromat 100x/1.46 Oil objective | Carl Zeiss | ||
| SYBR Gold | ThermoFischer | S11494 | cell permeant DNA stain |
| Zen Black 2012 software | Carl Zeiss | image acquisition and processing software |
Referenzen
- Kaufman, B. A., et al. The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures. Molecular Biology of the Cell. 18 (9), 3225-3236 (2007).
- Farge, G., et al. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A. Nature Communications. 3, 1013 (2012).
- Bogenhagen, D. F. Mitochondrial DNA nucleoid structure. Biochimica et Biophysica Acta. 1819 (9-10), 914-920 (2012).
- Kang, D., Kim, S. H., Hamasaki, N. Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions. Mitochondrion. 7 (1-2), 39-44 (2007).
- Tauber, J., et al. Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells. International Journal of Biochemistry & Cell Biology. 45 (3), 593-603 (2013).
- Garrido, N., et al. Composition and dynamics of human mitochondrial nucleoids. Molecular Biology of the Cell. 14 (4), 1583-1596 (2003).
- Pernas, L., Scorrano, L. Mito-Morphosis: Mitochondrial Fusion, Fission, and Cristae Remodeling as Key Mediators of Cellular Function. Annual Review of Physiology. 78, 505-531 (2016).
- Caielli, S., et al. Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus. Journal of Experimental Medicine. 213 (5), 697-713 (2016).
- Okayama, S., Ohta, K., Higashi, R., Nakamura, K. Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy. Microscopy (Oxf). 63 (Suppl 1), i35 (2014).
- Alan, L., Spacek, T., Jezek, P. Delaunay algorithm and principal component analysis for 3D visualization of mitochondrial DNA nucleoids by Biplane FPALM/dSTORM. European Biophysics Journal. 45 (5), 443-461 (2016).
- Kukat, C., et al. Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Proceedings of the National Academy of Sciences of the United States of America. 108 (33), 13534-13539 (2011).
- Kukat, C., Larsson, N. G. mtDNA makes a U-turn for the mitochondrial nucleoid. Trends in Cell Biology. 23 (9), 457-463 (2013).
- Betzig, E., et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 313 (5793), 1642-1645 (2006).
- Rust, M. J., Bates, M., Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods. 3 (10), 793-795 (2006).
- Benke, A., Manley, S. Live-cell dSTORM of cellular DNA based on direct DNA labeling. ChemBioChem. 13 (2), 298-301 (2012).
- Ishigaki, M., et al. STED super-resolution imaging of mitochondria labeled with TMRM in living cells. Mitochondrion. 28, 79-87 (2016).
- Waldchen, S., Lehmann, J., Klein, T., van de Linde, S., Sauer, M. Light-induced cell damage in live-cell super-resolution microscopy. Scientific Reports. 5, 15348 (2015).
- Gustafsson, M. G., et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophysical Journal. 94 (12), 4957-4970 (2008).
- Hirano, Y., Matsuda, A., Hiraoka, Y. Recent advancements in structured-illumination microscopy toward live-cell imaging. Microscopy (Oxf). 64 (4), 237-249 (2015).
- Brown, T. A., et al. Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Molecular and Cellular Biology. 31 (24), 4994-5010 (2011).
- Lewis, S. C., Uchiyama, L. F., Nunnari, J. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells. Science. 353 (6296), aaf5549 (2016).
- Dellinger, M., Geze, M. Detection of mitochondrial DNA in living animal cells with fluorescence microscopy. Journal of Microscopy. 204 (Pt 3), 196-202 (2001).
- Ban-Ishihara, R., Ishihara, T., Sasaki, N., Mihara, K., Ishihara, N. Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c. Proceedings of the National Academy of Sciences of the United States of America. 110 (29), 11863-11868 (2013).
- Zurek-Biesiada, D., et al. Localization microscopy of DNA in situ using Vybrant((R)) DyeCycle Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution. Experimental Cell Research. 343 (2), 97-106 (2016).
- He, J. Y., et al. The AAA(+) protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. Journal of Cell Biology. 176 (2), 141-146 (2007).
- Ashley, N., Harris, D., Poulton, J. Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining. Experimental Cell Research. 303 (2), 432-446 (2005).
- Jevtic, V., Kindle, P., Avilov, S. V. SYBR Gold dye enables preferential labeling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy. PLoS One. 13 (9), e0203956 (2018).
