Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy

Developers

Eligio F. Iannetti, Jan A. M. Smeitink, Peter H.G. M. Willems, Werner J. H. Koopman, etc.

Description of the technology

Mitochondria have a central role in cellular physiology and pathophysiology, and they display a highly variable morphology that is probably coupled to their functional state.

The technology allows unbiased and automated quantification of mitochondrial 'morphofunction' (i.e., morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living primary human skin fibroblasts.

Cells are cultured in 96-well plates and stained with tetramethyl rhodamine methyl ester, calcein-acetoxy-methyl ester and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors (i.e., relevant mitochondrial parameters such as fluorescence intensity and mitochondrial shape from the images). Subsequently, the descriptor data are subjected to a quality control algorithm based upon principal component analysis and interpreted using univariate, bivariate and multivariate analysis.

The protocol of the technology requires an operative time of ~4 h distributed over 2 d. Although the protocol is specifically developed for primary human skin fibroblasts, which are widely used in preclinical research, it can be adapted to other cell types and can be scaled up for implementation in high-content screening.

Practical application

The technology of analysis of mitochondrial morphofunction has high potential for fundamental science and medical practice. Mitochondria are cellular organelles that fulfill a wide range of functions in cellular metabolism. Functional defects in mitochondrial and/or cellular metabolism are often associated with aberrations in mitochondrial morphology and vice versa. These defects are not only observed in rare monogenic mitochondrial disorders, but are also associated with common pathologies such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, cancer, cardiac disease, diabetes, epilepsy and obesity. Moreover, a progressive decline in the expression of mitochondrial genes is a central feature of normal human aging. This means that maintaining normal mitochondrial morphofunction is of eminent importance for human health and, as a consequence, strategies for evaluation of this morphofunction are a subject of major interest in experimental research, pharmacological screening and clinical diagnostics. Thus, this technology can be highly valuable tool for study of many physiological and pathological processes mentioned above.

Laboratories

• Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Nijmegen (the Netherlands)
• Khondrion BV, Nijmegen (the Netherlands)
• Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboudumc, Nijmegen (the Netherlands)
• Centre for Systems Biology and Bioenergetics, Radboudumc, Nijmegen (the Netherlands)

Links

Publications

• Iannetti, E.F. et al. «Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy." 11 Nature Protocols (2016): 1693–1710.
• Iannetti, E. et al. «High-content and high-throughput analysis of mitochondrial dynamics." 63 Int. J. Biochem. Cell Biol. (2015): 66–70.
• Koopman, W.J.H. et al. «Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency." 289 Am. J. Physiol. Cell Physiol. (2005): C881-C890.