Tomáš Špaček

Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells

Mitochondrial DNA (mtDNA) is organized in nucleoids in complex with accessory proteins, proteins of mtDNA replication and gene expression machinery. A robust mtDNA genome is represented by hundreds to thousands of nucleoids in cell mitochondrion. Detailed information is lacking about the dynamics of nucleoid distribution within the mitochondrial network upon physiological and pathological events. Therefore, we used confocal microscopy to study mitochondrial nucleoid redistribution upon mitochondrial fission and following reintegration of the mitochondrial network. Fission was induced by oxidative stress at respiration inhibition by rotenone or upon elimination of the protonmotive force by uncoupling or upon canceling its electrical component, ΔΨ(m), by valinomycin; and by silencing of mitofusin MFN2. Agent withdrawal resulted in concomitant mitochondrial network reintegration. We found two major principal morphological states: (i) a tubular state of the mitochondrial network with equidistant nucleoid spacing, 1.10±0.2 nucleoids per μm, and (ii) a fragmented state of solitary spheroid objects in which several nucleoids were clustered. We rarely observed singular mitochondrial fragments with a single nucleoid inside and very seldom we observed empty fragments. Reintegration of fragments into the mitochondrial network re-established the tubular state with equidistant nucleoid spacing. The two major morphological states coexisted at intermediate stages. These observations suggest that both mitochondrial network fission and reconnection of the disintegrated network are nucleoid-centric, i.e., fission and new mitochondrial tubule formation are initiated around nucleoids. Analyses of combinations of these morphological icons thus provide a basis for a future mitochondrial morphology diagnostics.

Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β‐barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super‐resolution biplane fluorescence photoactivation localization microscopy with Eos‐conjugated, ICS‐located lactamase‐β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos‐mitochondrial fission protein‐1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MIC OS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ~20%. ATP synthase dimers vs. monomers and state‐3/state‐4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM‐detached cristae of OPA1‐ and mitofilin‐silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down‐regulation. Mitofilin‐depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.—Plecitá‐Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down‐regulation concomitant to MICOS clustering. FASEB J. 30, 1941–1957 (2016). www.fasebj.org

Mitochondrial nucleoid clusters protect newly synthesized mtDNA during Doxorubicin- and Ethidium Bromide-induced mitochondrial stress

Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.

Nkx6.1 decline accompanies mitochondrial DNA reduction but subtle nucleoid size decrease in pancreatic islet β-cells of diabetic Goto Kakizaki rats

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured “nucleoids” which were smaller by 25% (less diameters >150 nm) in GK β-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell nucleoid number (spatial density) was not observed, rejecting model of single mtDNA per nucleoid. The β-cell maintenance factor Nkx6.1 mRNA and protein were declining with age (>12-fold, 10 months) and decreasing with fasting hyperglycemia in GK rats, probably predetermining the impaired mtDNA replication (copy number decrease), while spatial expansion of mtDNA kept nucleoids with only smaller sizes than those containing much higher mtDNA in non-diabetic β-cells.

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

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 F1 subunit α (F1α). Obtained 3D images were analyzed with the help of Ripleys 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 F1α 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 F1α interdistances reflected cristae width decrease upon GSIS, since such repositioning of F1α 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.

Superoxide Generation, Bioenergetics Parameters, and Mitochondrial Morphology in Insulinoma INS-1E Cells upon Glucose Addition and ATPase Inhibitory Factor (IF1) Knockdown

We studied mitochondrial superoxide formation under conditions when the respiration substrates suddenly increase upon glucose addition, hence one might expect the elevated superoxide formation. We aimed to study this situation also while determining the concomitant bioenergetics responses. We have clearly demonstrated that mitochondria as the glucose sensor in pancreatic β-cells do decrease superoxide release to the matrix upon GSIS. To correlate resulting changes in surplus superoxide release to the matrix with bioenergetics parameters, we also inspected respiration and mitochondrial membrane potential ΔΨm. The employed 14 mM incremental glucose in cells cultivated with 11 mM glucose induced a small respiration elevation resulting in a slight non-significant increase in ratio of state 3/state 4 respiration. We further confirmed presence of the ATPase Inhibitory Factor 1, IF1, in pancreatic beta cells and studied its role in superoxide generation, regulation of bioenergetics parameters and mitochondrial architecture in INS1E cells.