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 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 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 MCL-1 inhibition by selective BH3 mimetics disrupts mitochondrial dynamics in iPSC-derived cardiomyocytes

2019 ◽  
Author(s):  
Megan L. Rasmussen ◽  
Nilay Taneja ◽  
Abigail C. Neininger ◽  
Lili Wang ◽  
Linzheng Shi ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Morphology ◽  
Outer Mitochondrial Membrane ◽  
Canonical Function ◽  
Bh3 Mimetics ◽  
Caspase Inhibition ◽  
Dynamics And Function ◽  
Apoptotic Activity ◽  
And Function

SummaryMCL-1 is a well characterized inhibitor of cell death that has also been shown to be a regulator of mitochondrial dynamics in human pluripotent stem cells (hPSCs). We used cardiomyocytes derived from hPSCs (hPSC-CMs) to uncover whether MCL-1 is crucial for cardiac function and survival. Inhibition of MCL-1 by BH3 mimetics, resulted in the disruption of mitochondrial morphology and dynamics as well as disorganization of the actin cytoskeleton. Interfering with MCL-1 function affects the homeostatic proximity of DRP-1 and MCL-1 at the outer mitochondrial membrane, resulting in decreased functionality of hPSC-CMs. BH3 mimetics targeting MCL-1 are promising anti-tumor therapeutics. Cardiomyocytes display abnormal functional cardiac performance even after caspase inhibition, supporting a non-apoptotic activity of MCL-1 in hPSC-CMs. Progression towards using BCL-2 family inhibitors, especially targeting MCL-1, depends on understanding not only its canonical function in preventing apoptosis, but also in the maintenance of mitochondrial dynamics and function.

scholarly journals Rapid purification and direct microassay of calbindin9kDa utilizing its solubility in perchloric acid

1993 ◽  
Vol 293 (1) ◽  
pp. 223-227 ◽  
Author(s):  
M J Hubbard
Keyword(s):  
Perchloric Acid ◽  
Calcium Binding ◽  
Cultured Cells ◽  
Peptide Bond ◽  
New Approach ◽  
Ef Hand ◽  
Rat Duodenum ◽  
Wet Weight ◽  
Old Rats ◽  
Rapid Purification

The 9 kDa calcium-binding protein, calbindin9kDa, was found to be soluble in 7% (v/v) perchloric acid. Calbindin9kDa was easily purified from rat duodenum in 1 day with perchloric acid precipitation followed by reverse-phase h.p.l.c. The yield was 21.4 +/- 2.3 nmol/g wet weight of tissue (mean +/- S.E.M.; n = 3) from normally fed 7-8-week-old rats (approx. 70% recovery). The purification was also effective with rabbit duodenum calbindin9kDa, but not with various other EF-hand calcium-binding proteins tested in the rat. Several criteria (h.p.l.c., u.v. spectrum, denaturing two-dimensional PAGE, N-terminal sequencing) indicated that the rat calbindin9kDa was purified to homogeneity and was not affected by proteolysis. High-affinity calcium-binding properties were retained and no evidence of isoforms or charge modification was observed. Residue 59, identified as Asn (not Asp as previously reported), was fully amidated. When adopted as a microassay with isocratic h.p.l.c., the perchloric acid procedure enabled rapid (less than 6 min) and direct (peptide bond absorbance) quantification of less than 1 pmol of calbindin9kDa. This new approach to purification and assay will be of particular utility for investigations of calbindin9kDa in previously intractable low-abundance sources (e.g. cultured cells).

scholarly journals Machine learning-based classification of mitochondrial morphology in primary neurons and brain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Garrett M. Fogo ◽  
Anthony R. Anzell ◽  
Kathleen J. Maheras ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Image Analysis ◽  
Cortical Neurons ◽  
Mitochondrial Dynamics ◽  
Automated Image Analysis ◽  
Mitochondrial Morphology ◽  
Analysis Pipeline ◽  
Genetic Ablation ◽  
Block Face

AbstractThe mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.

scholarly journals Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Anthony R. Anzell ◽  
Garrett M. Fogo ◽  
Zoya Gurm ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Ischemia Reperfusion Injury ◽  
Mitochondrial Dynamics ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fission ◽  
Conditional Knockout ◽  
Mitochondrial Morphology ◽  
Primary Neurons ◽  
Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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