Ultrastructural Features of Left Ventricle Cardiomyocytes in Preterm Newborn Rats.

The structure of left ventricular cardiomyocytes of 1 day preterm newborn rats was studied using transmission electron microscopy. It was shown that the relative area of the nucleus in cardiomyocytes of preterm rats is lower, and the relative area of the cytoplasm is higher than in full-term rats, while the relative areas of myofibrils and mitochondria do not differ. In cardiomyocytes of preterm rats damaged mitochondria, subsegmental myofibrillar contracture, and cytoplasmic swelling were found on the first postnatal day. Preterm birth in rats, in contrast to birth at term, is accompanied by the development of a number of ultrastructural damages in cardiomyocytes.

Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes.

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

Invariance of Mitochondria and Synapses in the Primary Visual Cortex of Mammals Provides Insight Into Energetics and Function.

The cerebral cortex accounts for substantial energy expenditure, primarily driven by the metabolic demands of synaptic signaling. Mitochondria, the organelles responsible for generating cellular energy, play a crucial role in this process. We investigated ultrastructural characteristics of the primary visual cortex in 18 phylogenetically diverse mammals, spanning a broad range of brain sizes from mouse to elephant. Our findings reveal remarkable uniformity in synapse density, postsynaptic density (PSD) length, and mitochondria density, indicating functional and metabolic constraints that maintain these fundamental features. Notably, we observed an average of 1.9 mitochondria per synapse across mammalian species. When considered together with the trend of decreasing neuron density with larger brain size, we find that brain enlargement in mammals is characterized by increasing proportions of synapses and mitochondria per cortical neuron. These results shed light on the adaptive mechanisms and metabolic dynamics that govern cortical ultrastructure across mammals.

Metallic and metallic oxide nanoparticles toxicity primarily targets the mitochondria of hepatocytes and renal cells.

Nanoparticles (NPs) are utilized in various applications, posing potential risks to human health, tissues, cells, and macromolecules. This study aimed to investigate the ultrastructural alterations in hepatocytes and renal tubular cells induced by metallic and metal oxide NPs. Adult healthy male Wistar albino rats (Rattus norvegicus) were divided into 6 (n = 7) control and 6 treated groups (n = 7). The rats in the treated groups exposed daily to silver NPs, gold NPs, zinc oxide NPs, silicon dioxide NPs, copper oxide NPs, and ferric oxide NPs for 35 days. The members of the control group for each corresponding NPs received the respective vehicle. Liver and kidney tissue blocks from all rats were processed for Transmission Electron Microscopy (TEM) examinations. The hepatocytes and renal tubular cells of all NPs-treated rats demonstrated mitochondrial ultrastructural alterations mainly cristolysis, swelling, membrane disruption, lucent matrices, matrices lysis, and electron-dense deposits. However, other organelles demonstrated injury but to a lesser extent in the form of shrunken nuclei, nuclear membrane indentation, endoplasmic reticulum fragmentation, cellular membranes enfolding, brush border microvilli disruption, lysosomal hyperplasia, ribosomes dropping, and peroxisome formation. One may conclude from the findings that the hepatocytes and the renal tubular cells mitochondria are the main targets for nanoparticles toxicity ending in mitochondrial disruption and cell injury. Further studies taking into account the relation of mitochondrial ultrastructural damage with a weakened antioxidant defense system induced by chronic exposure to nanomaterials are needed.

In situ structure and rotary states of mitochondrial ATP synthase in whole Polytomella cells.

Cells depend on a continuous supply of adenosine triphosphate (ATP), the universal energy currency. In mitochondria, ATP is produced by a series of redox reactions, whereby an electrochemical gradient is established across the inner mitochondrial membrane. The ATP synthase harnesses the energy of the gradient to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. We determined the structure of ATP synthase within mitochondria of the unicellular flagellate Polytomella by electron cryo-tomography and subtomogram averaging at up to 4.2-angstrom resolution, revealing six rotary positions of the central stalk, subclassified into 21 substates of the F1 head. The Polytomella ATP synthase forms helical arrays with multiple adjacent rows defining the cristae ridges. The structure of ATP synthase under native operating conditions in the presence of a membrane potential represents a pivotal step toward the analysis of membrane protein complexes in situ.

Mitochondrial-derived compartments are multilamellar domains that encase membrane cargo and cytosol.

Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here, we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains.

Leptin A deficiency affecting the mitochondrial dynamics of aged oocytes in medaka (Oryzias latipes).

Mitochondrial dysfunction and metabolic disorder have been associated to age-related subfertility, however, the precise molecular mechanism controlling the development of fertile oocytes in aging females remains elusive. Leptin plays an important role in the maintenance of energy homeostasis, as both excessive or insufficient levels can affect the body weight and fertility of mice. Here, we report that leptin A deficiency affects growth and shortens reproductive lifespan by reducing fertility in medaka (Oryzias latipes). Targeted disruption of lepa (lepa-/-) females reduced their egg laying and fertility compared to normal 3-month-old females (lepa+/+ sexual maturity), with symptoms worsening progressively at the age of 6 months and beyond. Transcriptomic analysis showed that differentially expressed genes involved in metabolic and mitochondrial pathways were significantly altered in lepa-/- ovaries compared with the normal ovaries at over 6 months old. The expression levels of the autophagy-promoting genes ulk1a, atg7 and atg12 were significantly differentiated between normal and lepa-/- ovaries, which were further confirmed by quantitative polymerase chain reaction analysis, indicating abnormal autophagy activation and mitochondrial dysfunction in oocyte development lacking lepa. Transmission electron microscopy observations further confirmed these mitochondrial disorders in lepa-deficient oocytes. In summary, these research findings provide novel insights into how leptin influences female fertility through mitochondrial-mediated oocyte development.

Analysis of beta-cell maturity and mitochondrial morphology in juvenile non-human primates exposed to maternal Western-style diet during development.

Introduction: Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. Methods: We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. Results: There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Discussion: Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

dldhcri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

Quantitative assessment of morphological changes in lipid droplets and lipid-mito interactions with aging in brown adipose.

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LDs) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. For example, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and the proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Our analyses showed reductions in LD volume, area, and perimeter in aged samples in comparison to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for the mitochondria interacting with LDs. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology with mitochondrial function, metabolism, and bioactivity in aged BAT.

Effects of vitrification on mitochondrial ultrastructure and membrane potential and its distribution in mouse oocytes.

BACKGROUND: Vitrification is commonly used for in vitro fertilization and has significant impact on gametes. OBJECTIVE: To investigate changes in ultrastructure, membrane potential and distribution of mitochondria in mouse oocytes after vitrification. MATERIALS AND METHODS: Mouse oocytes were divided into three groups: one group as fresh control, one group for the toxicity test (treated with cryoprotectant but without vitrification), and the other for vitrification. RESULTS: Most mitochondria in oocytes were damaged after cooling and warming, being rough and fuzzy in appearance, even swollen and broken. The membrane potential of the toxicity test group and the vitrification group was 0.320 +/-0.030 and 0.244 +/- 0.038, respectively, in comparison to the fresh group (0.398 +/- 0.043). The membrane potential of the vitrified oocytes was significantly lower than fresh oocytes and the toxicity test oocytes (P % 0.05), but there was no significant difference between fresh oocytes and the toxicity test oocytes (P > 0.05). Mitochondria in fresh oocytes were denser and strained stronger, with 59.5> distributed homogeneously and 36.4> polarized. The majority of mitochondria in the toxicity-tested oocytes were clustered (69.3>) and only a small portion were distributed homogeneously (19.6>), while mitochondria in vitrified oocytes were clustered (56.3>) and deficient (24.4>), and their fluorescent staining was weak and blurred. There was a significant disruption in mitochondrial function after vitrification. CONCLUSION: Vitrification alters the ultrastructure, membrane potential and distribution of mitochondria in oocytes, most likely caused by toxicity and mechanical injury. Doi.org/10.54680/fr24510110212.

Glucocorticoid Deflazacort Normalizes the Ultrastructure of Skeletal Muscles and the State of the Colon Microbiota in Dystrophin-Deficient Mice.

We studied the effect of enteral administration of the glucocorticoid deflazacort (DFC, 1.2 mg/kg per day, 28 days) on the state of skeletal muscles and tissue ultrastructure, as well as the composition of the colon microbiota in dystrophin-deficient mdx mice. DFC has been shown to reduce the intensity of degeneration/regeneration cycles in muscle fibers of mdx mice. This effect of DFC was accompanied by normalization of the size of sarcomeres of skeletal muscles of mdx mice, improvement of the ultrastructure of the subsarcolemmal population of mitochondria, and an increase in the number of organelles, as well as normalization of the number of contact interactions between the sarcoplasmic reticulum and mitochondria. In addition, DFC had a corrective effect on the colon microbiota of mdx mice, which manifested in an increase in the number of the Bifidobacterium genus microorganisms and a decrease in the level of E. coli with reduced enzymatic activity.

Sperm characteristics of the Telorchiidae (Digenea, Plagiorchioidea): First ultrastructural data on Telorchis attenuatus an intestinal parasite of Trachemys scripta elegans.

The ultrastructural features of the mature spermatozoon of Telorchis attenuatus (Digenea, Telorchiidae), an intestinal parasite of the red-eared turtle Trachemys scripta elegans (Testudines, Emydidae), are described using transmission electron microscopy (TEM). The mature spermatozoon of T. attenuatus is a filiform cell tapered at both ends and displays Bakhoum et al.'s type IV of digenean sperm cells. Spermatozoa of T. attenuatus have: (i) two axonemes of different lengths with the 9+'1' pattern of trepaxonematan Platyhelminthes, surrounded by a continuous submembranous layer of cortical microtubules at their anterior end, (ii) an external ornamentation of the plasma membrane following Quilichini et al.'s type 2 and associated with cortical microtubules, (iii) two bundles of parallel cortical microtubules with the maximum number situated in the anterior part of the sperm cell, (iv) spine-like bodies, (v) two mitochondria, and (vi) a large number of irregularly distributed glycogen granules. Furthermore, the morphology of the posterior spermatozoon extremity in T. attenuatus corresponds to the Quilichini et al.'s fasciolidean type. The results of the current study are especially compared to the existing information from other families within the superfamily Plagiorchioidea.

Vitamin D ameliorates particulate matter induced mitochondrial damages and calcium dyshomeostasis in BEAS-2B human bronchial epithelial cells.

BACKGROUND: Mitochondria is prone to oxidative damage by endogenous and exogenous sources of free radicals, including particulate matter (PM). Given the role of mitochondria in inflammatory disorders, such as asthma and chronic obstructive pulmonary disease, we hypothesized that supplementation of vitamin D may play a protective role in PM-induced mitochondrial oxidative damages of human bronchial epithelial BEAS-2B cells. METHODS: BEAS-2B cells were pretreated with 1,25(OH)2D3, an active form of vitamin D, for 1 h prior to 24-hour exposure to PM (SRM-1648a). Oxidative stress was measured by flow cytometry. Mitochondrial functions including mitochondrial membrane potential, ATP levels, and mitochondrial DNA copy number were analyzed. Additionally, mitochondrial ultrastructure was examined using transmission electron microscopy. Intracellular and mitochondrial calcium concentration changes were assessed using flow cytometry based on the expression of Fluo-4 AM and Rhod-2 AM, respectively. Pro-inflammatory cytokines, including IL-6 and MCP-1, were quantified using ELISA. The expression levels of antioxidants, including SOD1, SOD2, CAT, GSH, and NADPH, were determined. RESULTS: Our findings first showed that 24-hour exposure to PM led to the overproduction of reactive oxygen species (ROS) derived from mitochondria. PM-induced mitochondrial oxidation resulted in intracellular calcium accumulation, particularly within mitochondria, and alterations in mitochondrial morphology and functions. These changes included loss of mitochondrial membrane integrity, disarrayed cristae, mitochondrial membrane depolarization, reduced ATP production, and increased mitochondrial DNA copy number. Consequently, PM-induced mitochondrial damage triggered the release of certain inflammatory cytokines, such as IL-6 and MCP-1. Similar to the actions of mitochondrial ROS inhibitor MitoTEMPO, 1,25(OH)2D3 conferred protective effects on mtDNA alterations, mitochondrial damages, calcium dyshomeostasis, thereby decreasing the release of certain inflammatory cytokines. We found that greater cellular level of 1,25(OH)2D3 upregulated the expression of enzymatic (SOD1, SOD2, and CAT) and non-enzymatic (GSH and NADPH) antioxidants to modulate cellular redox homeostasis. CONCLUSION: Our study provides new evidence that 1,25(OH)2D3 acts as an antioxidant, enhancing BEAS-2B antioxidant responses to regulate mitochondrial ROS homeostasis and mitochondrial function, thereby enhancing epithelial defense against air pollution exposure.

Expansion microscopy reveals characteristic ultrastructural features of pathogenic budding yeast species.

Candida albicans is the most prevalent fungal pathogen associated with candidemia. Similar to other fungi, the complex life cycle of C. albicans has been challenging to study with high-resolution microscopy due to its small size. Here, we employed ultrastructure expansion microscopy (U-ExM) to directly visualise subcellular structures at high resolution in the yeast and during its transition to hyphal growth. N-hydroxysuccinimide (NHS)-ester pan-labelling in combination with immunofluorescence via snapshots of various mitotic stages provided a comprehensive map of nucleolar and mitochondrial segregation dynamics and enabled the resolution of the inner and outer plaque of spindle pole bodies (SPBs). Analyses of microtubules (MTs) and SPBs suggest that C. albicans displays a side-by-side SPB arrangement with a short mitotic spindle and longer astral MTs (aMTs) at the pre-anaphase stage. Modifications to the established U-ExM protocol enabled the expansion of six other human fungal pathogens, revealing that the side-by-side SPB configuration is a plausibly conserved feature shared by many fungal species. We highlight the power of U-ExM to investigate subcellular organisation at high resolution and low cost in poorly studied and medically relevant microbial pathogens.

Decreased skeletal muscle intramyocellular lipid droplet-mitochondrial contact contributes to myosteatosis in cancer cachexia.

Cancer cachexia, the unintentional loss of lean mass, contributes to functional dependency, poor treatment outcomes, and decreased survival. Although its pathogenicity is multifactorial, metabolic dysfunction remains a hallmark of cachexia. However, significant knowledge gaps exist in understanding the role of skeletal muscle lipid metabolism and dynamics in this condition. We examined skeletal muscle metabolic dysfunction, intramyocellular lipid droplet (LD) content, LD morphology and subcellular distribution, and LD-mitochondrial interactions using the Lewis lung carcinoma (LLC) murine model of cachexia. C57/BL6 male mice (n = 20) were implanted with LLC cells (106) in the right flank or underwent PBS sham injections. Skeletal muscle was excised for transmission electron microscopy (TEM; soleus), oil red O/lipid staining [tibialis anterior (TA)], and protein (gastrocnemius). LLC mice had a greater number (232%; P = 0.006) and size (130%; P = 0.023) of intramyocellular LDs further supported by increased oil-red O positive (87%; P = 0.0109) and "very high" oil-red O positive (178%; P = 0.0002) fibers compared with controls and this was inversely correlated with fiber size (R2 = 0.5294; P < 0.0001). Morphological analyses of LDs show increased elongation and complexity [aspect ratio: intermyofibrillar (IMF) = 9%, P = 0.046) with decreases in circularity [circularity: subsarcolemmal (SS) = 6%, P = 0.042] or roundness (roundness: whole = 10%, P = 0.033; IMF = 8%, P = 0.038) as well as decreased LD-mitochondria touch (-15%; P = 0.006), contact length (-38%; P = 0.036), and relative contact (86%; P = 0.004). Furthermore, dysregulation in lipid metabolism (adiponectin, CPT1b) and LD-associated proteins, perilipin-2 and perilipin-5, in cachectic muscle (P < 0.05) were observed. Collectively, we provide evidence that skeletal muscle myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in a preclinical model of cancer cachexia.NEW & NOTEWORTHY We sought to advance our understanding of skeletal muscle lipid metabolism and dynamics in cancer cachexia. Cachexia increased the number and size of intramyocellular lipid droplets (LDs). Furthermore, decreases in LD-mitochondrial touch, contact length, and relative contact along with increased LD shape complexity with decreases in circularity and roundness. Dysregulation in lipid metabolism and LD-associated proteins was also documented. Collectively, we show that myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in cancer cachexia.

Quantitative 3D electron microscopy characterization of mitochondrial structure, mitophagy, and organelle interactions in murine atrial fibrillation.

Atrial fibrillation (AF) is the most common clinical arrhythmia, however there is limited understanding of its pathophysiology including the cellular and ultrastructural changes rendered by the irregular rhythm, which limits pharmacological therapy development. Prior work has demonstrated the importance of reactive oxygen species (ROS) and mitochondrial dysfunction in the development of AF. Mitochondrial structure, interactions with other organelles such as sarcoplasmic reticulum (SR) and T-tubules (TT), and degradation of dysfunctional mitochondria via mitophagy are important processes to understand ultrastructural changes due to AF. However, most analysis of mitochondrial structure and interactome in AF has been limited to two-dimensional (2D) modalities such as transmission electron microscopy (EM), which does not fully visualize the morphological evolution of the mitochondria during mitophagy. Herein, we utilize focused ion beam-scanning electron microscopy (FIB-SEM) and perform reconstruction of three-dimensional (3D) EM from murine left atrial samples and measure the interactions of mitochondria with SR and TT. We developed a novel 3D quantitative analysis of FIB-SEM in a murine model of AF to quantify mitophagy stage, mitophagosome size in cardiomyocytes, and mitochondrial structural remodeling when compared with control mice. We show that in our murine model of spontaneous and continuous AF due to persistent late sodium current, left atrial cardiomyocytes have heterogenous mitochondria, with a significant number which are enlarged with increased elongation and structural complexity. Mitophagosomes in AF cardiomyocytes are located at Z-lines where they neighbor large, elongated mitochondria. Mitochondria in AF cardiomyocytes show increased organelle interaction, with 5X greater contact area with SR and are 4X as likely to interact with TT when compared to control. We show that mitophagy in AF cardiomyocytes involves 2.5X larger mitophagosomes that carry increased organelle contents. In conclusion, when oxidative stress overcomes compensatory mechanisms, mitophagy in AF faces a challenge of degrading bulky complex mitochondria, which may result in increased SR and TT contacts, perhaps allowing for mitochondrial Ca2+ maintenance and antioxidant production.

Mitochondrial Structure, Dynamics, and Physiology: Light Microscopy to Disentangle the Network.

Mitochondria serve as energetic and signaling hubs of the cell: This function results from the complex interplay between their structure, function, dynamics, interactions, and molecular organization. The ability to observe and quantify these properties often represents the puzzle piece critical for deciphering the mechanisms behind mitochondrial function and dysfunction. Fluorescence microscopy addresses this critical need and has become increasingly powerful with the advent of superresolution methods and context-sensitive fluorescent probes. In this review, we delve into advanced light microscopy methods and analyses for studying mitochondrial ultrastructure, dynamics, and physiology, and highlight notable discoveries they enabled.

Mitochondrial structural alterations in fibromyalgia: a pilot electron microscopy study.

OBJECTIVES: The pathogenesis of fibromyalgia (FM), characterised by chronic widespread pain and fatigue, remains notoriously elusive, hampering attempts to develop disease modifying treatments. Mitochondria are the headquarters of cellular energy metabolism, and their malfunction has been proposed to contribute to both FM and chronic fatigue. Thus, the aim of the current pilot study, was to detect structural changes in mitochondria of peripheral blood mononuclear cells (PBMCs) of FM patients, using transmission electron microscopy (TEM). METHODS: To detect structural mitochondrial alterations in FM, we analysed PBMCs from seven patients and seven healthy controls, using TEM. Patients were recruited from a specialised Fibromyalgia Clinic at a tertiary medical centre. After providing informed consent, participants completed questionnaires including the widespread pain index (WPI), symptoms severity score (SSS), fibromyalgia impact questionnaire (FIQ), beck depression inventory (BDI), and visual analogue scale (VAS), to verify a diagnosis of FM according to ACR criteria. Subsequently, blood samples were drawn and PBMCs were collected for EM analysis. RESULTS: TEM analysis of PBMCs showed several distinct mitochondrial cristae patterns, including total loss of cristae in FM patients. The number of mitochondria with intact cristae morphology was reduced in FM patients and the percentage of mitochondria that completely lacked cristae was increased. These results correlated with the WPI severity. Moreover, in the FM patient samples we observed a high percentage of cells containing electron dense aggregates, which are possibly ribosome aggregates. Cristae loss and possible ribosome aggregation were intercorrelated, and thus may represent reactions to a shared cellular stress condition. The changes in mitochondrial morphology suggest that mitochondrial dysfunction, resulting in inefficient oxidative phosphorylation and ATP production, metabolic and redox disorders, and increased reactive oxygen species (ROS) levels, may play a pathogenetic role in FM. CONCLUSIONS: We describe novel morphological changes in mitochondria of FM patients, including loss of mitochondrial cristae. While these observations cannot determine whether the changes are pathogenetic or represent an epiphenomenon, they highlight the possibility that mitochondrial malfunction may play a causative role in the cascade of events leading to chronic pain and fatigue in FM. Moreover, the results offer the possibility of utilising changes in mitochondrial morphology as an objective biomarker in FM. Further understanding the connection between FM and dysfunction of mitochondria physiology, may assist in developing both novel diagnostic tools as well as specific treatments for FM, such as approaches to improve/strengthen mitochondria function.

High-resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii.

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.