scholarly journals A comprehensive approach for artifact-free sample preparation and assessment of mitochondrial morphology in tissue and cultured cells

2021 ◽  
Author(s):  
Antentor Hinton ◽  
Prasanna Katti ◽  
Trace A. Christensen ◽  
Margaret Mungai ◽  
Jianqiang Shao ◽  
...  
Keyword(s):  
Structural Changes ◽  
Cultured Cells ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Morphology ◽  
Specimen Preparation ◽  
Preparation Methods ◽  
Cellular Environment ◽  
Stress Changes ◽  
Precise Relationship ◽  
And Function

Mitochondrial dynamics and morphology (fission, fusion, and the formation of nanotunnels) are very sensitive to the cellular environment and may be adversely affected by oxidative stress, changes in calcium levels, and hypoxia. Investigating the precise relationship between the organelle structure and function requires methods that can adequately preserve the structure while providing accurate, quantitative measurements of mitochondrial morphological attributes. Here, we demonstrate a practical approach for preserving and measuring fine structural changes in two-dimensional electron micrographs, obtained using transmission electron microscopy, highlighting the specific advantages of this technique. Additionally, this study defines a set of quantifiable metrics that can be applied to measure mitochondrial architecture and other organellar structures. Finally, we validated specimen preparation methods that avoid the introduction of morphological artifacts in mitochondrial appearance that do not require whole-animal perfusion.

scholarly journals Blocked O-GlcNAc cycling alters mitochondrial morphology, function, and mass

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elizabeth O. Akinbiyi ◽  
Lara K. Abramowitz ◽  
Brianna L. Bauer ◽  
Maria S. K. Stoll ◽  
Charles L. Hoppel ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Mitochondrial Morphology ◽  
Post Translational Modification ◽  
Mitochondrial Content ◽  
Protein Levels ◽  
Cellular Environment ◽  
And Function ◽  
The Impact

AbstractO-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.

Microscopy of porous ceramics

1989 ◽  
Vol 47 ◽  
pp. 438-439
Author(s):  
H. M. Kerch ◽  
R. A. Gerhardt
Keyword(s):  
Porous Ceramics ◽  
Specimen Preparation ◽  
High Porosity ◽  
Ion Milling ◽  
Forming Process ◽  
Preparation Methods ◽  
Pore Texture ◽  
Proper Design ◽  
And Function ◽  
Function Information

Highly porous ceramics are employed in a variety of engineering applications due to their unique mechanical, optical, and electrical characteristics. In order to achieve proper design and function, information about the pore structure must be obtained. Parameters of importance include pore size, pore volume, and size distribution, as well as pore texture and geometry. A quantitative determination of these features for high porosity materials by a microscopic technique is usually not done because artifacts introduced by either the sample preparation method or the image forming process of the microscope make interpretation difficult.Scanning electron microscopy for both fractured and polished surfaces has been utilized extensively for examining pore structures. However, there is uncertainty in distinguishing between topography and pores for the fractured specimen and sample pullout obscures the true morphology for samples that are polished. In addition, very small pores (nm range) cannot be resolved in the S.E.M. On the other hand, T.E.M. has better resolution but the specimen preparation methods involved such as powder dispersion, ion milling, and chemical etching may incur problems ranging from preferential widening of pores to partial or complete destruction of the pore network.

scholarly journals Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway

2004 ◽  
Vol 167 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Rebecca L. Frederick ◽  
J. Michael McCaffery ◽  
Kyle W. Cunningham ◽  
Koji Okamoto ◽  
Janet M. Shaw
Keyword(s):  
Cell Signaling ◽  
Calcium Binding ◽  
Cultured Cells ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Morphology ◽  
Genetic Studies ◽  
Ef Hand ◽  
Signaling Events ◽  
Apoptotic Activity ◽  
Mitochondrial Membrane Protein

Cell signaling events elicit changes in mitochondrial shape and activity. However, few mitochondrial proteins that interact with signaling pathways have been identified. Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs. We show that Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein. Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria. We demonstrate that Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics. Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function. Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells (Fransson et al., 2003. J. Biol. Chem. 278:6495–6502), Gem1p is not required for pheromone-induced yeast cell death. Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

scholarly journals Mitochondrial Perturbation Contributing to Cognitive Decline in Streptozotocin-Induced Type 1 Diabetic Rats

2018 ◽  
Vol 46 (4) ◽  
pp. 1668-1682 ◽  
Author(s):  
Yu Zhou ◽  
Siheng Lian ◽  
Jin Zhang ◽  
Donghai Lin ◽  
Caihua Huang ◽  
...  
Keyword(s):  
Cognitive Decline ◽  
Metabolic Pathways ◽  
Mitochondrial Dynamics ◽  
Diabetic Rats ◽  
Morris Water Maze Test ◽  
Mitochondrial Morphology ◽  
Underlying Mechanisms ◽  
And Function ◽  
Energy Failure

Background/Aims: Mitochondrial perturbation is a well-established cause of cognitive decline, but as yet it is unclear how mitochondria-associated neuronal abnormalities in type 1 diabetic (T1DM) brain contribute to cognitive decline. Methods: The streptozotocin (STZ)-induced mouse model of T1DM was used. The Morris water maze test was applied to assess the effect of T1DM on learning and memory. We detected changes in mitochondrial morphology, function and dynamics. Furthermore, we employed metabolomic analysis to reveal the underlying mechanisms of mitochondrial perturbation which contribute to cognitive decline. Results: Our results show that T1DM impairs mitochondrial dynamics, morphology and function in neurons, associated with a decline in cognitive ability. Metabolomic analyses revealed that T1DM mainly affects metabolic pathways involved in mitochondrial energy failure and impairs the antioxidative system. Conclusion: These results lay the basis for understanding the underlying mitochondria-associated causes of T1DM-associated cognitive decline and may provide a potential treatment strategy for this condition in future.

Morphological fluctuations of individual mitochondria in living cells

2021 ◽  
Author(s):  
Agustina Belén Fernández Casafuz ◽  
María Cecilia De Rossi ◽  
Luciana Bruno
Keyword(s):  
Network Architecture ◽  
Morphological Changes ◽  
Persistence Length ◽  
Mitochondrial Morphology ◽  
Directed Motion ◽  
Cellular Environment ◽  
Mitochondria Dynamics ◽  
Tension And Compression ◽  
Spatio Temporal ◽  
And Function

Abstract Uncovering the link between mitochondrial morphology, dynamics, positioning and function is challenging. Mitochondria are very flexible organelles that are subject to tension and compression within cells. Recent findings highlighted the importance of these mechanical aspects in the regulation of mitochondria dynamics, arising the question on which are the processes and mechanisms involved in their shape remodeling. In this work we explored in detail the morphological changes and spatio- temporal fluctuations of these organelles in living Xenopus laevis melanophores, a well- characterized cellular model. We developed an automatic method for the classification of mitochondria shapes based on the analysis of the curvature of the contour shape from confocal microscopy images. A persistence length of 2.1 μm was measured, quantifying, for the first time, the bending plasticity of mitochondria in their cellular environment. The shape evolution at the single organelle level was followed during a few minutes revealing that mitochondria can bend and unbend in the seconds timescale. Furthermore, the inspection of confocal movies simultaneously registering fluorescent mitochondria and microtubules suggests that the cytoskeleton network architecture and dynamics play a significant role in mitochondria shape remodeling and fluctuations. For instance changes from sinuous to elongated organelles related to transitions from confined behavior to fast directed motion along microtubule tracks were observed.

scholarly journals Mitochondrial Structural Changes in the Pathogenesis of Diabetic Retinopathy

2019 ◽  
Vol 8 (9) ◽  
pp. 1363 ◽  
Author(s):  
Roy ◽  
Kim ◽  
Sankaramoorthy
Keyword(s):  
Diabetic Retinopathy ◽  
High Glucose ◽  
Structural Changes ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Morphology ◽  
Mitochondrial Fragmentation ◽  
Diabetic Retina ◽  
High Glucose Condition ◽  
Physical Changes ◽  
Work Supports

Abstract: At the core of proper mitochondrial functionality is the maintenance of its structure and morphology. Physical changes in mitochondrial structure alter metabolic pathways inside mitochondria, affect mitochondrial turnover, disturb mitochondrial dynamics, and promote mitochondrial fragmentation, ultimately triggering apoptosis. In high glucose condition, increased mitochondrial fragmentation contributes to apoptotic death in retinal vascular and Müller cells. Although alterations in mitochondrial morphology have been detected in several diabetic tissues, it remains to be established in the vascular cells of the diabetic retina. From a mechanistic standpoint, our current work supports the notion that increased expression of fission genes and decreased expression of fusion genes are involved in promoting excessive mitochondrial fragmentation. While mechanistic insights are only beginning to reveal how high glucose alters mitochondrial morphology, the consequences are clearly seen as release of cytochrome c from fragmented mitochondria triggers apoptosis. Current findings raise the prospect of targeting excessive mitochondrial fragmentation as a potential therapeutic strategy for treatment of diabetic retinopathy. While biochemical and epigenetic changes have been reported to be associated with mitochondrial dysfunction, this review focuses on alterations in mitochondrial morphology, and their impact on mitochondrial function and pathogenesis of diabetic retinopathy.

scholarly journals Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 848
Author(s):  
Marco Coazzoli ◽  
Alessandra Napoli ◽  
Paulina Roux-Biejat ◽  
Clara De Palma ◽  
Claudia Moscheni ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Dynamics ◽  
Tumour Progression ◽  
Acid Sphingomyelinase ◽  
Mitochondrial Fusion ◽  
Mitochondrial Morphology ◽  
Transcriptional Level ◽  
Enhanced Expression ◽  
And Function ◽  
Stimulation Of

Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.

scholarly journals Acute exercise remodels mitochondrial membrane interactions in mouse skeletal muscle

2013 ◽  
Vol 115 (10) ◽  
pp. 1562-1571 ◽  
Author(s):  
Martin Picard ◽  
Benoit J. Gentil ◽  
Meagan J. McManus ◽  
Kathryn White ◽  
Kyle St. Louis ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Acute Exercise ◽  
Exercise Intervention ◽  
Cultured Cells ◽  
Mitochondrial Dynamics ◽  
Membrane Dynamics ◽  
Mitochondrial Morphology ◽  
Membrane Interactions

A unique property of mitochondria in mammalian cells is their ability to physically interact and undergo dynamic events of fusion/fission that remodel their morphology and possibly their function. In cultured cells, metabolic perturbations similar to those incurred during exercise influence mitochondrial fusion and fission processes, but it is unknown whether exercise acutely alters mitochondrial morphology and/or membrane interactions in vivo. To study this question, we subjected mice to a 3-h voluntarily exercise intervention following their normal physical activity patterns, and quantified mitochondrial morphology and membrane interactions in the soleus using a quantitative electron microscopy approach. A single exercise bout effectively decreased blood glucose ( P < 0.05) and intramyocellular lipid content ( P < 0.01), indicating increased muscle metabolic demand. The number of mitochondria spanning Z-lines and proportion of electron-dense contact sites (EDCS) between adjacent mitochondrial membranes were increased immediately after exercise among both subsarcolemmal (+116%, P < 0.05) and intermyofibrillar mitochondria (+191%, P < 0.001), indicating increased physical interactions. Mitochondrial morphology, and abundance of the mitochondrial pro-fusion proteins Mfn2 and OPA1 were unchanged. Collectively, these results support the notion that mitochondrial membrane dynamics are actively remodelled in skeletal muscle, which may be regulated by contractile activity and the metabolic state. Future studies are required to understand the implications of mitochondrial dynamics in skeletal muscle physiology during exercise and inactivity.

scholarly journals Viral Infection Modulates Mitochondrial Function

2021 ◽  
Vol 22 (8) ◽  
pp. 4260
Author(s):  
Xiaowen Li ◽  
Keke Wu ◽  
Sen Zeng ◽  
Feifan Zhao ◽  
Jindai Fan ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Death ◽  
Viral Infection ◽  
Viral Infections ◽  
Mitochondrial Dynamics ◽  
Innate Immune ◽  
Host Cells ◽  
Mitochondrial Morphology ◽  
And Function ◽  
Antiviral Role

Mitochondria are important organelles involved in metabolism and programmed cell death in eukaryotic cells. In addition, mitochondria are also closely related to the innate immunity of host cells against viruses. The abnormality of mitochondrial morphology and function might lead to a variety of diseases. A large number of studies have found that a variety of viral infections could change mitochondrial dynamics, mediate mitochondria-induced cell death, and alter the mitochondrial metabolic status and cellular innate immune response to maintain intracellular survival. Meanwhile, mitochondria can also play an antiviral role during viral infection, thereby protecting the host. Therefore, mitochondria play an important role in the interaction between the host and the virus. Herein, we summarize how viral infections affect microbial pathogenesis by altering mitochondrial morphology and function and how viruses escape the host immune response.

scholarly journals FOXO3a regulates BNIP3 and modulates mitochondrial calcium, dynamics, and function in cardiac stress

2016 ◽  
Vol 311 (6) ◽  
pp. H1540-H1559 ◽  
Author(s):  
Antoine H. Chaanine ◽  
Erik Kohlbrenner ◽  
Scott I. Gamb ◽  
Adam J. Guenzel ◽  
Katherine Klaus ◽  
...  
Keyword(s):  
Heart Failure ◽  
Mitochondrial Dynamics ◽  
Left Ventricular ◽  
Attractive Potential ◽  
Mitochondrial Morphology ◽  
Cardiac Stress ◽  
Virus Serotype ◽  
Dynamics And Function ◽  
And Function

The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca2+, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca2+ cycling, Ca2+ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/role-of-foxo3a-in-heart-failure/ .

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