3D super-resolution microscopy reflects mitochondrial cristae alternations and mtDNA nucleoid size and distribution

Abstract

3D super-resolution microscopy based on the direct stochastic optical reconstruction microscopy (dSTORM) with primary Alexa-Fluor-647-conjugated antibodies is a powerful method for accessing changes of objects that could be normally resolved only by electron microscopy. Despite the fact that mitochondrial cristae yet to become resolved, we have indicated changes in cristae width and/or morphology by dSTORM of ATP-synthase F subunit α (Fα). 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. In fasting state of model pancreatic β-cells, INS-1E, MFD between Fα were ~80 nm at 0 and 3 mM glucose, whereas decreased to 61 nm and 57 nm upon glucose-stimulated insulin secretion (GSIS) at 11 mM and 20 mM glucose, respectively. Shorter Fα interdistances reflected cristae width decrease upon GSIS, since such repositioning of Fα correlated to average 20 nm and 15 nm cristae width at 0 and 3 mM glucose, and 9 nm or 8 nm after higher glucose simulating GSIS (11, 20 mM glucose, respectively). Also, submitochondrial entities such as nucleoids of mtDNA were resolved e.g. after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM. MFD in distances distribution histograms reflected an average nucleoid diameter (<100 nm) and average distances between nucleoids (~1000 nm). Double channel PALM/dSTORM with Eos-lactamase-β plus anti-TFAM dSTORM confirmed the latter average inter-nucleoid distance. In conclusion, 3D single molecule (dSTORM) microscopy is a reasonable tool for studying mitochondrion.

The Functional Impact of Mitochondrial Structure Across Subcellular Scales

“…As a result, cristae have traditionally been thought of as stable structures despite the widespread observations of individual mitochondria as dynamic structures over the past two decades (Nunnari et al, 1997;Bleazard et al, 1999;Karbowski et al, 2004;Szabadkai et al, 2004;Twig et al, 2008b;Molina et al, 2009;Lewis et al, 2016). However, recent technological advances have brought the resolution of the light microscope down into the tens of nanometer range which has finally permitted observation of cristae structures in live cells (Dlasková et al, 2018(Dlasková et al, , 2019Ježek and Dlasková, 2019;Jakobs et al, 2020; Figure 1 bottom left). Application of these super-resolution microscopy techniques to mitochondria from several types of cell culture systems have indeed revealed that cristae are dynamic structures capable of both fission and fusion-type events similar to those which occur at the organelle level (Huang et al, 2018;Stephan et al, 2019;Wang et al, 2019).…”

Oral pyruvate prevents glaucomatous neurodegeneration

“…We previously demonstrated PARP upregulation in RGCs during D2 glaucoma and PARP can inhibit glycolysis by impacting hexokinase (5). Elevated glucose is associated with poor outcomes in brain injury and disease (12,13), and high glucose concentrations lead to decreased mitochondrial cristae and mitochondrial remodeling in diabetic and cell models (14,15). D2 RGC mitochondria have decreased cristae at corresponding disease time points (10).…”

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+Ratio

“…PIs were isolated from C57BL/6J mice (or when indicated from C57BL/6N mice) and used immediately for measurements. Nevertheless, they could be maintained for up to 1 week in the transient culture as previously described (17,19,60).…”