Age is proposed to be associated with altered structure and function of mitochondria; however, in fully-differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel-by-pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during 'flickers' of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly-elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly-motile. Mitochondria that are motile are located more peripherally in the cell than non-motile mitochondria.

Eukaryotic cells attempt to maintain an optimal size, resulting in size homeostasis. While cellular content scales isometrically with cell size, allometric laws indicate that metabolism per mass unit should decline with increasing size. Here we use elutriation and single-cell flow cytometry to analyze mitochondrial scaling with cell size. While mitochondrial content increases linearly, mitochondrial membrane potential and oxidative phosphorylation are highest at intermediate cell sizes. Thus, mitochondrial content and functional scaling are uncoupled. The nonlinearity of mitochondrial functionality is cell size, not cell cycle, dependent, and it results in an optimal cell size whereby cellular fitness and proliferative capacity are maximized. While optimal cell size is controlled by growth factor signaling, its establishment and maintenance requires mitochondrial dynamics, which can be controlled by the mevalonate pathway. Thus, optimization of cellular fitness and functionality through mitochondria can explain the requirement for size control, as well as provide means for its maintenance.

Evidence from models and experiments suggests that the networked structure observed in mitochondria emerges at the critical point of a phase transition controlled by fission and fusion rates. If mitochondria are poised at criticality, the relevant network quantities should scale with the system's size. However, whether or not the expected finite-size effects take place has not been demonstrated yet. Here, we first provide a theoretical framework to interpret the scaling behavior of mitochondrial network quantities by analyzing two conceptually different models of mitochondrial dynamics. Then, we perform a finite-size scaling analysis of real mitochondrial networks extracted from microscopy images and obtain scaling exponents comparable with critical exponents from models and theory. Overall, we provide a universal description of the structural phase transition in mammalian mitochondria.

In pigs, mitochondrial DNA (mtDNA) polymorphism and the correlation to reproductive performance across breeds and individuals have been largely reported, however, experimental proof has never been provided. In this study, we analyzed 807 sows for correlation of total number born (TNB) and mitotype, which presented the maximum of 1.73 piglets for mtDNA contribution. Cybrid models representing different mitotypes were generated for identification of the mtDNA effect. Results indicated significant differences on cellular and molecular characteristics among cybrids, including energy metabolic traits, mtDNA copy numbers and transcriptions, mRNA and protein expressions on mitochondrial biogenesis genes and reproduction-related genes. Referring to mitotypes, the cybrids with prolific mitotypes presented significantly higher oxygen consumption rate (OCR) productions, mtDNA transcriptions and copy numbers than those with common mitotypes, while both mRNA and protein expressions of PPARA, TFAM, ER1, ER2, and ESRRG in prolific cybrids were significantly higher than those with common mitotypes. Cybrid models reflected the mtDNA effect on pig litter size, suggesting the potential application of mtDNA polymorphism in pig selection and breeding practices.

With a combined carrier frequency of 1:200, heteroplasmic mitochondrial DNA (mtDNA) mutations cause human disease in ∼1:5000 of the population. Rapid shifts in the level of heteroplasmy seen within a single generation contribute to the wide range in the severity of clinical phenotypes seen in families transmitting mtDNA disease, consistent with a genetic bottleneck during transmission. Although preliminary evidence from human pedigrees points towards a random drift process underlying the shifting heteroplasmy, some reports describe differences in segregation pattern between different mtDNA mutations. However, based on limited observations and with no direct comparisons, it is not clear whether these observations simply reflect pedigree ascertainment and publication bias. To address this issue, we studied 577 mother-child pairs transmitting the m.11778G>A, m.3460G>A, m.8344A>G, m.8993T>G/C and m.3243A>G mtDNA mutations. Our analysis controlled for inter-assay differences, inter-laboratory variation and ascertainment bias. We found no evidence of selection during transmission but show that different mtDNA mutations segregate at different rates in human pedigrees. m.8993T>G/C segregated significantly faster than m.11778G>A, m.8344A>G and m.3243A>G, consistent with a tighter mtDNA genetic bottleneck in m.8993T>G/C pedigrees. Our observations support the existence of different genetic bottlenecks primarily determined by the underlying mtDNA mutation, explaining the different inheritance patterns observed in human pedigrees transmitting pathogenic mtDNA mutations.

Insulin resistance, a critical component of type 2 diabetes (T2D), precedes and predicts T2D onset. T2D is also associated with mitochondrial dysfunction. To define the cause-effect relationship between insulin resistance and mitochondrial dysfunction, we compared mitochondrial metabolism in induced pluripotent stem cells (iPSC) from 5 healthy individuals and 4 patients with genetic insulin resistance due to insulin receptor mutations. Insulin-resistant iPSC had increased mitochondrial number and decreased mitochondrial size. Mitochondrial oxidative function was impaired, with decreased citrate synthase activity and spare respiratory capacity. Simultaneously, expression of multiple glycolytic enzymes was decreased, while lactate production increased 80%. These perturbations were accompanied by an increase in ADP/ATP ratio and 3-fold increase in AMPK activity, indicating energetic stress. Insulin-resistant iPSC also showed reduced catalase activity and increased susceptibility to oxidative stress. Thus, insulin resistance can lead to mitochondrial dysfunction with reduced mitochondrial size, oxidative activity, and energy production.

To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell-cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear-encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

Mitochondrial dynamic process is important for cell viability, metabolic activity, and mitochondria health. Here, we present a protocol for measuring mitochondrial size through immunofluorescence staining, confocal imaging, and analysis in ImageJ. We describe the steps for tissue processing, antigen retrieval, mitochondrial staining using an integrating immunofluorescence assay, and computerized image analysis to measure each mitochondrial size in mouse and human liver tissues. This protocol reduces tissue sample volume and processing time for the preparation of primary cells. For complete details on the use and execution of this protocol, please refer to Pearah et al.1.

Arcidae, comprising about 260 species of ark shells, is an ecologically and economically important lineage of bivalve mollusks. Interestingly, mitochondrial genomes of several Arcidae species are 2-3 times larger than those of most bilaterians, and are among the largest bilaterian mitochondrial genomes reported to date. The large mitochondrial genome size is mainly due to expansion of unassigned regions (regions that are functionally unassigned). Previous work on unassigned regions of Arcidae mtDNA genomes has focused on nucleotide-level analyses to observe sequence characteristics, however the origin of expansion remains unclear.

Plant mitochondrial genomes have excessive size relative to coding capacity, a low mutation rate in genes and a high rearrangement rate. They also have abundant non-tandem repeats often including pairs of large repeats which cause isomerization of the genome by recombination, and numerous repeats of up to several hundred base pairs that recombine only when the genome is stressed by DNA damaging agents or mutations in DNA repair pathway genes. Early work on mitochondrial genomes led to the suggestion that repeats in the size range from several hundred to a few thousand base pair are underrepresented. The repeats themselves are not well-conserved between species, and are not always annotated in mitochondrial sequence assemblies. We systematically identified and compared these repeats, which are important clues to mechanisms of DNA maintenance in mitochondria. We developed a tool to find and curate non-tandem repeats larger than 50bp and analyzed the complete mitochondrial sequences from 157 plant species. We observed an interesting difference between taxa: the repeats are larger and more frequent in the vascular plants. Analysis of closely related species also shows that plant mitochondrial genomes evolve in dramatic bursts of breakage and rejoining, complete with DNA sequence gain and loss. We suggest an adaptive explanation for the existence of the repeats and their evolution.

To explore mechanisms by which SGLT2 inhibitors protect diabetic hearts from heart failure, we examined the effect of empagliflozin (Empa) on the ultrastructure of cardiomyocytes in the noninfarcted region of the diabetic heart after myocardial infarction (MI). OLETF, a rat model of type 2 diabetes, and its nondiabetic control, LETO, received a sham operation or left coronary artery ligation 12 h before tissue sampling. Tissues were sampled from the posterior ventricle (i.e., the remote noninfarcted region in rats with MI). The number of mitochondria was larger and small mitochondria were more prevalent in OLETF than in LETO. Fis1 expression level was higher in OLETF than in LETO, while phospho-Ser637-Drp1, total Drp1, Mfn1/2, and OPA1 levels were comparable. MI further reduced the size of mitochondria with increased Drp1-Ser616 phosphorylation in OLETF. The number of autophagic vacuoles was unchanged after MI in LETO but was decreased in OLETF. Lipid droplets in cardiomyocytes and tissue triglycerides were increased in OLETF. Empa administration (10 mg/kg per day) reduced blood glucose and triglycerides and paradoxically increased lipid droplets in cardiomyocytes in OLETF. Empa suppressed Fis1 upregulation, increased Bnip3 expression, and prevented reduction in both mitochondrial size and autophagic vacuole number after MI in OLETF. Together with the results of our parallel study showing upregulation of SOD2 and catalase by Empa, the results indicate that Empa normalizes the size and number of mitochondria in diabetic hearts and that diabetes-induced excessive reduction in mitochondrial size after MI was prevented by Empa via suppression of ROS and restoration of autophagy.

The cellular content of mitochondria changes dynamically during development and in response to external stimuli, but the underlying mechanisms remain obscure. To systematically identify molecular probes and pathways that control mitochondrial abundance, we developed a high-throughput imaging assay that tracks both the per cell mitochondrial content and the cell size in confluent human umbilical vein endothelial cells. We screened 28,786 small molecules and observed that hundreds of small molecules are capable of increasing or decreasing the cellular content of mitochondria in a manner proportionate to cell size, revealing stereotyped control of these parameters. However, only a handful of compounds dissociate this relationship. We focus on one such compound, BRD6897, and demonstrate through secondary assays that it increases the cellular content of mitochondria as evidenced by fluorescence microscopy, mitochondrial protein content, and respiration, even after rigorous correction for cell size, cell volume, or total protein content. BRD6897 increases uncoupled respiration 1.6-fold in two different, non-dividing cell types. Based on electron microscopy, BRD6897 does not alter the percent of cytoplasmic area occupied by mitochondria, but instead, induces a striking increase in the electron density of existing mitochondria. The mechanism is independent of known transcriptional programs and is likely to be related to a blockade in the turnover of mitochondrial proteins. At present the molecular target of BRD6897 remains to be elucidated, but if identified, could reveal an important additional mechanism that governs mitochondrial biogenesis and turnover.

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 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 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.

Proliferation of selfish genetic elements has led to significant genome size expansion in plastid and mitochondrial genomes of various eukaryotic lineages. Within the red algae, such expansion events are only known in the plastid genomes of the Proteorhodophytina, a highly diverse group of mesophilic microalgae. By contrast, they have never been described in the much understudied red algal mitochondrial genomes. Therefore, it remains unclear how widespread such organellar genome expansion events are in this eukaryotic phylum. Here, we describe new mitochondrial and plastid genomes from 25 red algal species, thereby substantially expanding the amount of organellar sequence data available, especially for Proteorhodophytina, and show that genome expansions are common in this group. We confirm that large plastid genomes are limited to the classes Rhodellophyceae and Porphyridiophyceae, which, in part, are caused by lineage-specific expansion events. Independently expanded mitochondrial genomes-up to three times larger than typical red algal mitogenomes-occur across Proteorhodophytina classes and a large shift toward high GC content occurred in the Stylonematophyceae. Although intron proliferation is the main cause of plastid and mitochondrial genome expansion in red algae, we do not observe recent intron transfer between different organelles. Phylogenomic analyses of mitochondrial and plastid genes from our expanded taxon sampling yielded well-resolved phylogenies of red algae with strong support for the monophyly of Proteorhodophytina. Our work shows that organellar genomes followed different evolutionary dynamics across red algal lineages.

Organelle genome studies of Fabaceae, an economically and ecologically important plant family, have been biased towards the plastid genome (plastome). Thus far, less than 15 mitochondrial genome (mitogenome) sequences of Fabaceae have been published, all but four of which belong to the subfamily Papilionoideae, limiting the understanding of size variation and content across the family. To address this, four mitogenomes were sequenced and assembled from three different subfamilies (Cercidoideae, Detarioideae and Caesalpinioideae).

In this study, the complete mitochondrial (mt) genomes of Chrysoporthe austroafricana (190,834 bp), C. cubensis (89,084 bp) and C. deuterocubensis (124,412 bp) were determined. Additionally, the mitochondrial genome of another member of the Cryphonectriaceae, namely Cryphonectria parasitica (158,902 bp), was retrieved and annotated for comparative purposes. These genomes showed high levels of synteny, especially in regions including genes involved in oxidative phosphorylation and electron transfer, unique open reading frames (uORFs), ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as intron positions. Comparative analyses revealed signatures of duplication events, intron number and length variation, and varying intronic ORFs which highlighted the genetic diversity of mt genomes among the Cryphonectriaceae. These mt genomes showed remarkable size polymorphism. The size polymorphism in the mt genomes of these closely related Chrysoporthe species was attributed to the varying number and length of introns, coding sequences and to a lesser extent, intergenic sequences. Compared to publicly available fungal mt genomes, the C. austroafricana mt genome is the second largest in the Ascomycetes thus far.

Mitochondrial genomes (mitogenomes) are remarkably diverse in genome size and organization, but the origins of dynamic mitogenome architectures are still poorly understood. For instance, the mutational burden hypothesis postulates that the drastic difference between large plant mitogenomes and streamlined animal mitogenomes can be driven by their different mutation rates. However, inconsistent trends between mitogenome sizes and mutation rates have been documented in several lineages. These conflicting results highlight the need of systematic and sophisticated investigations on the evolution and diversity of mitogenome architecture. This study took advantage of the strikingly variable mitogenome size among different yeast species and also among intraspecific strains, examined sequence dynamics of introns, GC-clusters, tandem repeats, mononucleotide repeats (homopolymers) and evaluated their contributions to genome size variation. The contributions of these sequence features to mitogenomic variation are dependent on the timescale, over which extant genomes evolved from their last common ancestor, perhaps due to a combination of different turnover rates of mobile sequences, variable insertion spaces, and functional constraints. We observed a positive correlation between mitogenome size and the level of genetic drift, suggesting that mitogenome expansion in yeast is likely driven by multiple types of sequence insertions in a primarily nonadaptive manner. Although these cannot be explained directly by the mutational burden hypothesis, our results support an important role of genetic drift in the evolution of yeast mitogenomes.

We present the complete mitochondrial genomes (mitogenomes) of Trisidos kiyoni and Potiarca pilula, both important species from the family Arcidae (Arcoida: Arcacea). Typical bivalve mtDNA features were described, such as the relatively conserved gene number (36 and 37), a high A + T content (62.73% and 61.16%), the preference for A + T-rich codons, and the evidence of non-optimal codon usage. The mitogenomes of Arcidae species are exceptional for their extraordinarily large and variable sizes and substantial gene rearrangements. The mitogenome of T. kiyoni (19,614 bp) and P. pilula (28,470 bp) are the two smallest Arcidae mitogenomes. The compact mitogenomes are weakly associated with gene number and primarily reflect shrinkage of the non-coding regions. The varied size in Arcidae mitogenomes reflect a dynamic history of expansion. A significant positive correlation is observed between mitogenome size and the combined length of cox1-3, the lengths of Cytb, and the combined length of rRNAs (rrnS and rrnL) (P < 0.001). Both protein coding genes (PCGs) and tRNA rearrangements is observed in P. pilula and T. kiyoni mitogenomes. This analysis imply that the complicated gene rearrangement in mitochondrial genome could be considered as one of key characters in inferring higher-level phylogenetic relationship of Arcidae.

The order Alismatales is a hotspot for evolution of plant mitochondrial genomes characterized by remarkable differences in genome size, substitution rates, RNA editing, retrotranscription, gene loss and intron loss. Here we have sequenced the complete mitogenomes of Zostera marina and Stratiotes aloides, which together with previously sequenced mitogenomes from Butomus and Spirodela, provide new evolutionary evidence of genome size reduction, gene loss and transfer to the nucleus. The Zostera mitogenome includes a large portion of DNA transferred from the plastome, yet it is the smallest known mitogenome from a non-parasitic plant. Using a broad sample of the Alismatales, the evolutionary history of ribosomal protein gene loss is analyzed. In Zostera almost all ribosomal protein genes are lost from the mitogenome, but only some can be found in the nucleus.

Nuclear mitochondrial DNA segment (Numt) insertion describes a well-known phenomenon of mitochondrial DNA transfer into a eukaryotic nuclear genome. However, it has not been well understood, especially in plants. Numt insertion patterns vary from species to species in different kingdoms. In this study, the patterns were surveyed in nine plant species, and we found some tip-offs. First, when the mitochondrial genome size is relatively large, the portion of the longer Numt is also larger than the short one. Second, the whole genome duplication event increases the ratio of the shorter Numt portion in the size distribution. Third, Numt insertions are enriched in exon regions. This analysis may be helpful for understanding plant evolution.