3D super-resolution dSTORM microscopy reflects morphology changes in mitochondrial cristae and mtDNA nucleoids P. Ježek1*, A. Dlasková1, H. Engstová, L. Plecitá - Hlavatá, K. Smolková, J. Špačková, V. Pavluch, T. Špaček, 1Institute of Physiology of the Czech Academy of Sciences *Corresponding Author
The direct stochastic optical reconstruction microscopy (dSTORM) possesses an advantage in the ability to resolve structures and protein proximities smaller than the average diameter of the mitochondrial reticulum tubules (typically 300-400 nm). One of the yet unexplored entities of mitochondrion are cristae  and nucleoids, the confined sites of mitochondrial DNA (mtDNA) in complex with proteins . Despite the unresolved cristae, narrowing in cristae width and changes in cristae outlets (disassembly of mitofilin clusters) were indicated by dSTORM of ATP-synthase F1 subunit alpha and mitofilin, respectively, in hypoxia-adapted HepG2 cells after the enhanced substrate load (4,4'-dimethyl-2-oxoglutarate, dm2OG, addition). Obtained 3D images were analyzed with the help of Ripley's K-function modeling spatial patterns or transferring them into distance distribution function. Resulting histograms of distances frequency distribution provide most frequent distances (MFD) between the localized single antibody molecules. The observed changes correlated with cristae width narrowing in transmission electron microscopy images and enhanced fraction of the ATP-synthase dimers in BN-PAGE. MFD analysis turned out to be useful also for mtDNA nucleoids resolved after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM, or when using TFAM dSTORM . MFD in distances distribution histograms reflected an average nucleoid diameter (<100 nm) and average distances between nucleoids (~1000 nm).
References  Plecitá-Hlavatá L, et al. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering. FASEB J., 2016 30:1941-1957  Alán, L., Špaček, T., & Ježek, P. Delaunay algorithm and principal component analysis for 3D visualization of mitochondrial DNA nucleoids by Biplane FPALM/dSTORM. Eur. Biophys. J. 45, 443-461 (2016).  Plecitá-Hlavatá L, Dlasková A, Engstová H, Špaček T, Kahancová A, Pavluch V, Smolková K, Špačková J, Ježek P. 3D super-resolution microscopy reflects mitochondrial cristae and mtDNA nucleoid morphology changes. Biochim. Biophys. Acta special issue EBEC 2018
Funding by GACR grant No.16-04788S
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