scholarly journals Leaf Mesophyll Mitochondrial Polarization Assessment in Arabidopsis thaliana

2021 ◽  
Vol 4 (4) ◽  
pp. 84
Author(s):  
Cesar Flores-Herrera ◽  
Emilia R. Gutiérrez-Mireles ◽  
Manuel Gutiérrez-Aguilar
Keyword(s):  
Arabidopsis Thaliana ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Fluorescent Protein ◽  
Mitochondrial Morphology ◽  
Plant Leaves ◽  
Abiotic Stressors ◽  
Model Plant Arabidopsis Thaliana ◽  
Plant Arabidopsis Thaliana

Plant leaves present an intricate array of layers providing a robust barrier against pathogens and abiotic stressors. However, these layers may also constitute an obstacle for the assessment of intracellular processes, especially when using fluorescence microscopy approaches. Current methods for leaf mitochondrial membrane potential determinations have been traditionally performed in thin mesophyll sections, in isolated protoplasts or in fluorescent protein-expressing transgenic plants. This may limit the amount of information obtained about overall mitochondrial morphology in intact leaves. Here, we detail a fast and straightforward protocol to assess changes in leaf mitochondrial membrane potential associated with mitochondrial dysfunction in the model plant Arabidopsis thaliana. This protocol also permits mitochondrial shape, dynamics and polarity assessment in leaves subjected to diverse stress conditions.

scholarly journals Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

1996 ◽  
Vol 44 (12) ◽  
pp. 1363-1372 ◽  
Author(s):  
M Poot ◽  
Y Z Zhang ◽  
J A Krämer ◽  
K S Wells ◽  
L J Jones ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Epifluorescence Microscopy ◽  
Mitochondrial Morphology ◽  
Microtubule Network ◽  
Multiple Features ◽  
Permeabilized Cells ◽  
Dye Staining ◽  
And Function

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Metformin Collaborates With PINK1/Mfn2 Overexpression Prevented Isoproterenol-Induced Cardiomyocyte Injury By Improving Mitochondrial Function

2021 ◽  
Author(s):  
Zhuang Ma ◽  
Zuheng Liu ◽  
Yuting Xue ◽  
Hao Zhang ◽  
Wenjun Xiong ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Energy Metabolism ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Mitochondrial Morphology ◽  
Dependent Manner ◽  
Combination Strategy ◽  
Mitochondrial Energy Metabolism ◽  
Atp Production

Abstract Background: Both mitochondrial quality control and energy metabolism are critical in maintaining the physiological function of cardiomyocytes. Previous studies indicated that PGC-1α is a transcription co-activator in promoting mitochondrial energy metabolism which would be beneficial for cardiomyocytes. However, PGC-1α overexpression in heart tissues could also result in the development of cardiomyopathy. This discrepancy in vivo and in vitro might be due to neglecting the elimination of damaged mitochondrial. Thus, an integration strategy of mitochondrial biogenesis and mitophagy might be beneficial.Methods: We studied the function of PINK1 in mitophagy in isoproterenol (Iso)-induced cardiomyocyte injury. Adenovirus was used to provoke an overexpression of the PINK1/Mfn2 protein. Mitochondrial morphology was examined via electron microscopy and confocal microscopy. Cardiomyocytes injury were measured by mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and apoptosis. Metformin was used to increase mitochondrial biogenesis, the level of which was detected via immunoblotting. Additionally, mitochondrial respiratory function was measured by ATP production and oxygen consumption rate (OCR). Results: Cardiomyocytes treated with Iso had high levels of PINK1 and low levels of Mfn2 in a time-dependent manner. PINK1 overexpression promoted mitophagy, alleviated Iso-induced reduction in MMP, reduced ROS production and the apoptotic rate. In addition to increasing mitophagy, metformin could promote mitochondrial biogenensis and the overexpression of Mfn2 induce mitochondrial fusion. Moreover, metformin treatment and PINK1/Mfn2 overexpression reduced the mitochondrial dysfunction by inhibiting the generation of ROS, and leading to an increase in both ATP production and mitochondrial membrane potential in Iso-induced cardiomyocytes injury. Conclusion: Our findings indicate that a combination strategy may help ameliorate myocardial injury through mitophagy and mitochondrial biogenesis.

scholarly journals Protein Kinase Cδ Targets Mitochondria, Alters Mitochondrial Membrane Potential, and Induces Apoptosis in Normal and Neoplastic Keratinocytes When Overexpressed by an Adenoviral Vector

1999 ◽  
Vol 19 (12) ◽  
pp. 8547-8558 ◽  
Author(s):  
Luowei Li ◽  
Patricia S. Lorenzo ◽  
Krisztina Bogi ◽  
Peter M. Blumberg ◽  
Stuart H. Yuspa
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Protein Kinase ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Fluorescent Protein ◽  
Protein Fusion ◽  
Cell Death Pathway ◽  
Morphological Criteria ◽  
Protein Kinase Cδ

ABSTRACT Inactivation of protein kinase Cδ (PKCδ) is associated with resistance to terminal cell death in epidermal tumor cells, suggesting that activation of PKCδ in normal epidermis may be a component of a cell death pathway. To test this hypothesis, we constructed an adenovirus vector carrying an epitope-tagged PKCδ under a cytomegalovirus promoter to overexpress PKCδ in normal and neoplastic keratinocytes. While PKCδ overexpression was detected by immunoblotting in keratinocytes, the expression level of other PKC isozymes, including PKCα, PKCɛ, PKCζ, and PKCη, did not change. Calcium-independent PKC-specific kinase activity increased after infection of keratinocytes with the PKCδ adenovirus. Activation of PKCδ by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a nanomolar concentration was lethal to normal and neoplastic mouse and human keratinocytes overexpressing PKCδ. Lethality was inhibited by PKC selective inhibitors, GF109203X and Ro-32-0432. TPA-induced cell death was apoptotic as evidenced by morphological criteria, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, DNA fragmentation, and increased caspase activity. Subcellular fractionation indicated that PKCδ translocated to a mitochondrial enriched fraction after TPA activation, and this finding was confirmed by confocal microscopy of cells expressing a transfected PKCδ-green fluorescent protein fusion protein. Furthermore, activation of PKCδ in keratinocytes altered mitochondrial membrane potential, as indicated by rhodamine-123 fluorescence. Mitochondrial inhibitors, rotenone and antimycin A, reduced TPA-induced cell death in PKCδ-overexpressing keratinocytes. These results indicate that PKCδ can initiate a death pathway in keratinocytes that involves direct interaction with mitochondria and alterations of mitochondrial function.

scholarly journals Manganese (II) chloride leads to dopaminergic neurotoxicity by promoting mitophagy through BNIP3-mediated oxidative stress in SH-SY5Y cells

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Yanning Huang ◽  
Qiaolin Wen ◽  
Jinfeng Huang ◽  
Man Luo ◽  
Yousheng Xiao ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Clinical Manifestations ◽  
Receptor Protein ◽  
Ros Generation ◽  
Mitochondrial Morphology ◽  
Human Neuroblastoma ◽  
Protein Levels ◽  
Mitochondrial Membrane Potential Loss

Abstract Background Manganese overexposure can induce neurotoxicity, lead to manganism and result in clinical manifestations similar to those of parkinsonism. However, the underlying molecular mechanism is still unclear. This study demonstrated that MnCl2 induces mitophagy and leads to neurotoxicity by promoting BNIP3-mediated reactive oxygen species (ROS) generation. Methods Human neuroblastoma SH-SY5Y cells were used throughout our experiments. Cell viability was detected by cell proliferation/toxicity test kits. Mitochondrial membrane potential was measured by flow cytometry. ROS generation was detected using a microplate reader. Protein levels were evaluated by Western blot. Transmission electron microscopy was used to evaluate mitochondrial morphology. Co-immunoprecipitation was used to verify the interaction between BNIP3 and LC3. Results MnCl2 led to loss of mitochondrial membrane potential and apoptosis of SH-SY5Y cells by enhancing expression of BNIP3 and conversion of LC3-I to LC3-II. Moreover, MnCl2 reduced expression of the mitochondrial marker protein TOMM20 and promoted interaction between BNIP3 and LC3. The results also indicated that a decrease in BNIP3 expression reduced the mitochondrial membrane potential loss, attenuated apoptosis and reduced mitochondrial autophagosome formation in SH-SY5Y cells after MnCl2 treatment. Finally, we found that manganese-induced ROS generation could be reversed by the antioxidant N-acetyl cysteine (NAC) or silencing BNIP3 expression. Conclusions BNIP3 mediates MnCl2-induced mitophagy and neurotoxicity in dopaminergic SH-SY5Y cells through ROS. Thus, BNIP3 contributes to manganese-induced neurotoxicity by functioning as a mitophagy receptor protein.

scholarly journals Regulation of mitochondrial morphology and function by O-GlcNAcylation in neonatal cardiac myocytes

2011 ◽  
Vol 300 (6) ◽  
pp. R1296-R1302 ◽  
Author(s):  
Ayako Makino ◽  
Jorge Suarez ◽  
Thomas Gawlowski ◽  
Wenlong Han ◽  
Hong Wang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cardiac Myocytes ◽  
Mitochondrial Membrane Potential ◽  
High Glucose ◽  
Mitochondrial Membrane ◽  
Control Level ◽  
Mitochondrial Morphology ◽  
Cell Physiology ◽  
Mitochondrial Fragmentation ◽  
Complex Iv

Mitochondria are crucial organelles in cell life serving as a source of energy production and as regulators of Ca2+ homeostasis, apoptosis, and development. Mitochondria frequently change their shape by fusion and fission, and recent research on these morphological dynamics of mitochondria has highlighted their role in normal cell physiology and disease. In this study, we investigated the effect of high glucose on mitochondrial dynamics in neonatal cardiac myocytes (NCMs). High-glucose treatment of NCMs significantly decreased the level of optical atrophy 1 (OPA1) (mitochondrial fusion-related protein) protein expression. NCMs exhibit two different kinds of mitochondrial structure: round shape around the nuclear area and elongated tubular structures in the pseudopod area. High-glucose-treated NCMs exhibited augmented mitochondrial fragmentation in the pseudopod area. This effect was significantly decreased by OPA1 overexpression. High-glucose exposure also led to increased O-GlcNAcylation of OPA1 in NCMs. GlcNAcase (GCA) overexpression in high-glucose-treated NCMs decreased OPA1 protein O-GlcNAcylation and significantly increased mitochondrial elongation. In addition to the morphological change caused by high glucose, we observed that high glucose decreased mitochondrial membrane potential and complex IV activity and that OPA1 overexpression increased both levels to the control level. These data suggest that decreased OPA1 protein level and increased O-GlcNAcylation of OPA1 protein by high glucose lead to mitochondrial dysfunction by increasing mitochondrial fragmentation, decreasing mitochondrial membrane potential, and attenuating the activity of mitochondrial complex IV, and that overexpression of OPA1 and GCA in cardiac myocytes may help improve the cardiac dysfunction in diabetes.

scholarly journals Goa1p of Candida albicans Localizes to the Mitochondria during Stress and Is Required for Mitochondrial Function and Virulence

2009 ◽  
Vol 8 (11) ◽  
pp. 1706-1720 ◽  
Author(s):  
Adrienne Bambach ◽  
Mariana P. Fernandes ◽  
Anup Ghosh ◽  
Michael Kruppa ◽  
Deepu Alex ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Membrane Potential ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Fluorescent Protein ◽  
Human Neutrophils ◽  
Agar Media ◽  
Green Fluorescent

ABSTRACT Using a Tn7 transposon library of Candida albicans, we have identified a mutant that exhibited sensitivity in drop plate assays to oxidants such as menadione and hydrogen peroxide. To verify the role of the mutated gene in stress adaptation, null mutants were constructed and phenotypically characterized. Because of its apparent functions in growth and oxidant adaptation, we have named the gene GOA1. Goa1p appears to be unique to the CTG subclade of the Saccharomycotina, including C. albicans. Mutants of C. albicans lacking goa1 (strain GOA31) were more sensitive to 6 mM H2O2 and 0.125 mM menadione than the wild type (wt) or a gene-reconstituted (GOA32) strain. The sensitivity to oxidants correlated with reduced survival of the GOA31 mutant in human neutrophils and avirulence compared to control strains. Other phenotypes of GOA31 include reduced growth and filamentation in 10% serum, Spider, and SLAD agar media and an inability to form chlamydospores. Since Goa1p has an N-terminal mitochondrion localization site, we also show that green fluorescent protein-tagged Goa1p shows a mitochondrionlike distribution during oxidant or osmotic stress. Further, the inability of GOA31 to grow in medium containing lactate, ethanol, or glycerol as the sole carbon source indicates that the mitochondria are defective in the mutant. To determine how Goa1p contributes to mitochondrial function, we compared the wt, GOA32, and GOA31 strains for mitochondrial electrical membrane potential, respiration, and oxidative phosphorylation. We now show that GOA31, but not the wt or GOA32, had decreased respiration and mitochondrial membrane potential such that mutant cells are unable to drive oxidative phosphorylation. This is the first report in C. albicans of a respiratory defect caused by a loss of mitochondrial membrane potential.

scholarly journals Chemical-Free Extraction of Functional Mitochondria Using a Microfluidic Device

Inventions ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. 68 ◽  
Author(s):  
Yu-Han Hsiao ◽  
Ching-Wen Li ◽  
Jui-Chih Chang ◽  
Sung-Tzu Chen ◽  
Chin-San Liu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Microfluidic Device ◽  
Complex I ◽  
Structural Damage ◽  
Mitochondrial Membrane ◽  
Normal Activity ◽  
Mitochondrial Morphology ◽  
Transmission Electron ◽  
Device Extraction

This paper proposes the use of a chip-based microfluidic device to extract functional and chemical free mitochondria. A simple microfluidic device was designed and fabricated. An osteosarcoma cybrid cell line was employed to demonstrate the efficiency of the proposed microfluidic device. The membrane proteins (mitochondrial complex I-V and Tom20) and morphology of the extracted mitochondria were examined by Western blot and transmission electron microscopy (TEM), respectively. The purity and mitochondrial membrane potential of the extracted mitochondria were individually measured by 10-N-alkyl acridine orange and tetramethylrhodamine ethyl ester staining via flow cytometry. Experimental results revealed that expressed pattern of complex I–V in device-extracted mitochondria was close to that of mitochondria in total cell lysis and device extraction significantly prevented chemical modification of complex IV protein via a conventional kit, although device extract similar amounts of mitochondria to the conventional kit revealed by Tom20 expression. Furthermore, purity of device-extracted mitochondria was above 93.7% and mitochondria still retained normal activity after device extraction proven by expression of mitochondrial membrane potential as well as the entire mitochondrial morphology. These results confirmed that the proposed microfluidic device could obtain functional mitochondria without structural damage.

scholarly journals Arabidopsis thaliana Embryo Sac Mitochondrial Membrane Potential Stain

BIO-PROTOCOL ◽  
2014 ◽  
Vol 4 (10) ◽  
Author(s):  
María Martin ◽  
Diego Fiol ◽  
Eduardo Zabaleta ◽  
Gabriela Pagnussat
Keyword(s):  
Arabidopsis Thaliana ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Embryo Sac

scholarly journals Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion

1999 ◽  
Vol 276 (3) ◽  
pp. C611-C620 ◽  
Author(s):  
Jorge Daniel Cortese
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Binding Proteins ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The variety of mitochondrial morphology in healthy and diseased cells can be explained by regulated mitochondrial fusion. Previously, a mitochondrial outer membrane fraction containing fusogenic, aluminum fluoride (AlF4)-sensitive GTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese, Exp. Cell Res. 240: 122–133, 1998). Quantitative confocal microscopy now reveals that mtg transiently increases mitochondrial membrane potential (ΔΨ) when added to permeabilized rat hepatocytes (15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large mtg-induced ΔΨ increment is blocked by fusogenic GTPase-specific modulators such as guanosine 5′- O-(3-thiotriphosphate), excess GTP (>100 μM), and AlF4, suggesting a linkage between ΔΨ and mitochondrial fusion. Accordingly, stereometric analysis shows that decreasing ΔΨ or ATP synthesis with respiratory inhibitors limits mtg- and AlF4-induced mitochondrial fusion. Also, a specific G protein inhibitor ( Bordetella pertussis toxin) hyperpolarizes mitochondria and leads to a loss of AlF4-dependent mitochondrial fusion. These results place mtg-induced ΔΨ changes upstream of AlF4-induced mitochondrial fusion, suggesting that GTPases exert ΔΨ-dependent control of the fusion process. Mammalian mitochondrial morphology thus can be modulated by cellular energetics.

scholarly journals Il ruolo di TAF12B e UVR3 nel ciclo circadiano dei vegetali

2013 ◽  
Author(s):  
Susanna Pollastri
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Bioinformatic Analysis ◽  
Model Organisms ◽  
Cell Level ◽  
Circadian Oscillators ◽  
Model Plant Arabidopsis Thaliana ◽  
Green Fluorescent ◽  
Plant Arabidopsis Thaliana

The book goes over the studies carried out on the circadian oscillators in plants, performed using the model plant Arabidopsis thaliana. The plant's oscillators seem to include similar components to those described for other model organisms. A bioinformatic analysis on the Arabidopsis genome found the genes TAF12b, At1g17440 and UVR3, At3g15620. Both the genes were cloned with the GFP green fluorescent protein in order to locate this protein at cell level. Furthermore, Arabidopsis SALK line plants were grown and selected, mutating the genes in question, and in parallel gene expression tests were performed on the main circadian rhythm genes in order to monitor their expression both in conditions of alternating light and dark, and in constant conditions. From the data that has emerged to date it seems that both genes are involved in the Arabidopsis's circadian rhythms.

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