scholarly journals Identification of the Bok Interactome Using Proximity Labeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Laura M. Szczesniak ◽  
Caden G. Bonzerato ◽  
Richard J. H. Wojcikiewicz
Keyword(s):  
Family Member ◽  
Transmembrane Domain ◽  
Mitochondrial Fission ◽  
Family Members ◽  
Potential Interaction ◽  
Mitochondrial Morphology ◽  
Mitochondrial Fragmentation ◽  
Knock Out ◽  
Inositol 1,4,5 Trisphosphate ◽  
Apoptosis Regulation

The function of the Bcl-2 family member Bok is currently enigmatic, with various disparate roles reported, including mediation of apoptosis, regulation of mitochondrial morphology, binding to inositol 1,4,5-trisphosphate receptors, and regulation of uridine metabolism. To better define the roles of Bok, we examined its interactome using TurboID-mediated proximity labeling in HeLa cells, in which Bok knock-out leads to mitochondrial fragmentation and Bok overexpression leads to apoptosis. Labeling with TurboID-Bok revealed that Bok was proximal to a wide array of proteins, particularly those involved in mitochondrial fission (e.g., Drp1), endoplasmic reticulum-plasma membrane junctions (e.g., Stim1), and surprisingly among the Bcl-2 family members, just Mcl-1. Comparison with TurboID-Mcl-1 and TurboID-Bak revealed that the three Bcl-2 family member interactomes were largely independent, but with some overlap that likely identifies key interactors. Interestingly, when overexpressed, Mcl-1 and Bok interact physically and functionally, in a manner that depends upon the transmembrane domain of Bok. Overall, this work shows that the Bok interactome is different from those of Mcl-1 and Bak, identifies novel proximities and potential interaction points for Bcl-2 family members, and suggests that Bok may regulate mitochondrial fission via Mcl-1 and Drp1.

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.

scholarly journals Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 450 ◽  
Author(s):  
Takeshi Tokuyama ◽  
Asei Hirai ◽  
Isshin Shiiba ◽  
Naoki Ito ◽  
Keigo Matsuno ◽  
...  
Keyword(s):  
Mitochondrial Disease ◽  
Mitochondrial Myopathy ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Critical Role ◽  
Leigh Syndrome ◽  
Mitochondrial Morphology ◽  
Mitochondrial Fragmentation ◽  
Cellular Toxicity ◽  
Morphologic Changes

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.

scholarly journals Fzo1, a Protein Involved in Mitochondrial Fusion, Inhibits Apoptosis

2004 ◽  
Vol 279 (50) ◽  
pp. 52726-52734 ◽  
Author(s):  
Rie Sugioka ◽  
Shigeomi Shimizu ◽  
Yoshihide Tsujimoto
Keyword(s):  
Cell Death ◽  
Conformational Changes ◽  
Apoptotic Cell ◽  
Mitochondrial Fission ◽  
Apoptotic Cell Death ◽  
Mitochondrial Fusion ◽  
Mitochondrial Morphology ◽  
Human Homolog ◽  
Mitochondrial Fragmentation ◽  
Apoptotic Death

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochromecrelease and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.

scholarly journals Porphyromonas gingivalis infection promotes mitochondrial dysfunction through Drp1-dependent mitochondrial fission in endothelial cells

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Tong Xu ◽  
Qin Dong ◽  
Yuxiao Luo ◽  
Yanqing Liu ◽  
Liang Gao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mitochondrial Dysfunction ◽  
Porphyromonas Gingivalis ◽  
Vascular Endothelial Cells ◽  
Mitochondrial Fission ◽  
Mitochondrial Morphology ◽  
Mitochondrial Fragmentation ◽  
Atp Production ◽  
Luminescence Assay ◽  
The Impact

AbstractPorphyromonas gingivalis (P. gingivalis), a key pathogen in periodontitis, has been shown to accelerate the progression of atherosclerosis (AS). However, the definite mechanisms remain elusive. Emerging evidence supports an association between mitochondrial dysfunction and AS. In our study, the impact of P. gingivalis on mitochondrial dysfunction and the potential mechanism were investigated. The mitochondrial morphology of EA.hy926 cells infected with P. gingivalis was assessed by transmission electron microscopy, mitochondrial staining, and quantitative analysis of the mitochondrial network. Fluorescence staining and flow cytometry analysis were performed to determine mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP) levels. Cellular ATP production was examined by a luminescence assay kit. The expression of key fusion and fission proteins was evaluated by western blot and immunofluorescence. Mdivi-1, a specific Drp1 inhibitor, was used to elucidate the role of Drp1 in mitochondrial dysfunction. Our findings showed that P. gingivalis infection induced mitochondrial fragmentation, increased the mtROS levels, and decreased the MMP and ATP concentration in vascular endothelial cells. We observed upregulation of Drp1 (Ser616) phosphorylation and translocation of Drp1 to mitochondria. Mdivi-1 blocked the mitochondrial fragmentation and dysfunction induced by P. gingivalis. Collectively, these results revealed that P. gingivalis infection promoted mitochondrial fragmentation and dysfunction, which was dependent on Drp1. Mitochondrial dysfunction may represent the mechanism by which P. gingivalis exacerbates atherosclerotic lesions.

scholarly journals Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

2004 ◽  
Vol 15 (11) ◽  
pp. 5001-5011 ◽  
Author(s):  
Yang-ja Lee ◽  
Seon-Yong Jeong ◽  
Mariusz Karbowski ◽  
Carolyn L. Smith ◽  
Richard J. Youle
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Fission ◽  
Mitochondrial Morphology ◽  
Apoptosis Induction ◽  
Wild Type ◽  
Mitochondrial Fragmentation ◽  
Down Regulation ◽  
Multiple Components ◽  
Mitochondrial Morphogenesis ◽  
Short Hairpin Rnas

During apoptosis, the mitochondrial network fragments. Using short hairpin RNAs for RNA interference, we manipulated the expression levels of the proteins hFis1, Drp1, and Opa1 that are involved in mitochondrial fission and fusion in mammalian cells, and we characterized their functions in mitochondrial morphology and apoptosis. Down-regulation of hFis1 powerfully inhibits cell death to an extent significantly greater than down-regulation of Drp1 and at a stage of apoptosis distinct from that induced by Drp1 inhibition. Cells depleted of Opa1 are extremely sensitive to exogenous apoptosis induction, and some die spontaneously by a process that requires hFis1 expression. Wild-type Opa1 may function normally as an antiapoptotic protein, keeping spontaneous apoptosis in check. However, if hFis1 is down-regulated, cells do not require Opa1 to prevent apoptosis, suggesting that Opa1 may be normally counteracting the proapoptotic action of hFis1. We also demonstrate in this study that mitochondrial fragmentation per se does not result in apoptosis. However, we provide further evidence that multiple components of the mitochondrial morphogenesis machinery can positively and negatively regulate apoptosis.

scholarly journals Downregulation of Drp1 and Fis1 Inhibits Mitochondrial Fission and Prevents High Glucose-Induced Apoptosis in Retinal Endothelial Cells

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1662
Author(s):  
Dongjoon Kim ◽  
Aravind Sankaramoorthy ◽  
Sayon Roy
Keyword(s):  
Endothelial Cells ◽  
Diabetic Retinopathy ◽  
High Glucose ◽  
Cell Apoptosis ◽  
Mitochondrial Fission ◽  
Confocal Imaging ◽  
Mitochondrial Morphology ◽  
Mitochondrial Fragmentation ◽  
Vascular Cell ◽  
Retinal Endothelial Cells

Diabetic retinopathy is a prevalent microvascular complication characterized by apoptotic vascular cell loss in the retina. Previous studies have shown that high glucose (HG)-induced mitochondrial fragmentation plays a critical role in promoting retinal vascular cell apoptosis. Here, we investigated whether downregulation of mitochondrial fission genes, Fis1 and Drp1, which are overexpressed in HG condition, prevents mitochondrial fragmentation, preserves mitochondrial function, and protects retinal endothelial cells from apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (5 mM glucose) or HG (30 mM glucose) medium; in parallel, cells grown in HG medium were transfected with either Fis1 siRNA or Drp1 siRNA, or both siRNAs in combination, or scrambled siRNA as control. Live-cell confocal imaging showed decreased mitochondrial fission in cells transfected with Fis1 siRNA or Drp1 siRNA concomitant with reduced TUNEL-positive cells and a decrease in the expression of pro-apoptotic proteins, Bax and cleaved caspase 3, under HG condition. Importantly, the combined siRNA approach against Fis1 and Drp1 prevented HG-induced changes in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The findings from this study indicate that reducing HG-induced overexpression of mitochondrial fission genes preserves mitochondrial morphology and prevents retinal vascular cell apoptosis associated with diabetic retinopathy.

scholarly journals Dynamin-related protein 1 mediates low glucose-induced endothelial dysfunction in human arterioles

2017 ◽  
Vol 312 (3) ◽  
pp. H515-H527 ◽  
Author(s):  
Michael J. Tanner ◽  
Jingli Wang ◽  
Rong Ying ◽  
Tisha B. Suboc ◽  
Mobin Malik ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Endothelial Function ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Cardiovascular Complications ◽  
Mitochondrial Morphology ◽  
Vascular Relaxation ◽  
Mitochondrial Fragmentation ◽  
Low Glucose

Intensive glycemic regulation has resulted in an increased incidence of hypoglycemia. Hypoglycemic burden correlates with adverse cardiovascular complications and contributes acutely and chronically to endothelial dysfunction. Prior data indicate that mitochondrial dysfunction contributes to hypoglycemia-induced endothelial dysfunction, but the mechanisms behind this linkage remain unknown. We attempt to determine whether clinically relevant low-glucose (LG) exposures acutely induce endothelial dysfunction through activation of the mitochondrial fission process. Characterization of mitochondrial morphology was carried out in cultured endothelial cells by using confocal microscopy. Isolated human arterioles were used to explore the effect LG-induced mitochondrial fission has on the formation of detrimental reactive oxygen species (ROS), bioavailability of nitric oxide (NO), and endothelial-dependent vascular relaxation. Fluorescence microscopy was employed to visualize changes in mitochondrial ROS and NO levels and videomicroscopy applied to measure vasodilation response. Pharmacological disruption of the profission protein Drp1 with Mdivi-1 during LG exposure reduced mitochondrial fragmentation among vascular endothelial cells (LG: 0.469; LG+Mdivi-1: 0.276; P = 0.003), prevented formation of vascular ROS (LG: 2.036; LG+Mdivi-1: 1.774; P = 0.005), increased the presence of NO (LG: 1.352; LG+Mdivi-1: 1.502; P = 0.048), and improved vascular dilation response to acetylcholine (LG: 31.6%; LG+Mdivi-1; 78.5% at maximum dose; P < 0.001). Additionally, decreased expression of Drp1 via siRNA knockdown during LG conditions also improved vascular relaxation. Exposure to LG imparts endothelial dysfunction coupled with altered mitochondrial phenotypes among isolated human arterioles. Disruption of Drp1 and subsequent mitochondrial fragmentation events prevents impaired vascular dilation, restores mitochondrial phenotype, and implicates mitochondrial fission as a primary mediator of LG-induced endothelial dysfunction. NEW & NOTEWORTHY Acute low-glucose exposure induces mitochondrial fragmentation in endothelial cells via Drp1 and is associated with impaired endothelial function in human arterioles. Targeting of Drp1 prevents fragmentation, improves vasofunction, and may provide a therapeutic target for improving cardiovascular complications among diabetics. Listen to this article’s corresponding podcast @ http://ajpheart.podbean.com/e/mitochondrial-dynamics-impact-endothelial-function/ .

Abstract 093: Adrenergic Stimulation Induces Mitochondrial Fragmentation and Cell Injury through PKD1-dependent Phosphorylation of DLP1 in H9c2 Cardiac Myoblasts.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Bong Sook Jhun ◽  
Jin O-Uchi ◽  
Stephen Hurst ◽  
Shey-Shing Sheu
Keyword(s):  
Protein Kinase ◽  
Mitochondrial Dysfunction ◽  
Cell Injury ◽  
Mitochondrial Fission ◽  
Dominant Negative ◽  
Mitochondrial Morphology ◽  
Catecholamine Level ◽  
Adrenergic Stimulation ◽  
Mitochondrial Fragmentation ◽  
Cardiac Myoblasts

Introduction: Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiac myocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed in various pathophysiological states of human heart such as heart failure, where the catecholamine level is elevated. However, it is still unclear what kinds of cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions. Hypothesis: Adrenergic signaling induces cardiac mitochondrial morphology changes and mitochondrial dysfunction, which simultaneously contribute to cardiac injury. Methods: H9c2 cardiac myoblasts were stimulated by α 1 -adrenoceptor (α 1 -AR) agonist phenylephrine and mitochondrial morphology was monitored by confocal microscopy. Translocation and phosphorylation of a mitochondrial fission protein, dynamin-like protein 1 (DLP1) was observed from whole cell lysates, cytosolic proteins and mitochondrial proteins by western blotting. Results: We found that persistent α 1 -AR stimulation induced mitochondrial fragmentation, followed by an increase in the production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria to the cytosol in H9c2 cardiac myoblasts. These effects were abolished by the treatment of α 1 -AR antagonist, prazosin. Further, mitochondrial fragmentation by α 1 -AR stimulation was inhibited by expression of the dominant-negative fission mutant DLP1-K38A, suggesting that the mitochondrial fission is required for mitochondrial fragmentation observed in α 1 -AR stimulation. We also found that DLP1 was translocated from cytosol to mitochondria under α 1 -AR stimulation. In addition, activation of protein kinase D1 (PKD1), a protein kinase downstream of α 1 -AR signaling, led to the phosphorylation of DLP1 at serine 637 which lies within a putative PKD phosphorylation consensus motif. Conclusion: α 1 -AR signaling induces mitochondrial fragmentation and cell injury, possibly through PKD1-dependent phosphorylation of DLP1.

scholarly journals Targeting Dynamin 2 as a Novel Pathway to Inhibit Cardiomyocyte Apoptosis Following Oxidative Stress

2016 ◽  
Vol 39 (6) ◽  
pp. 2121-2134 ◽  
Author(s):  
Danchen Gao ◽  
Jian Yang ◽  
Yutao Wu ◽  
Qiwen Wang ◽  
Qiaoling Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fission ◽  
Mitochondrial Morphology ◽  
Ros Production ◽  
Protective Effects ◽  
Mitochondrial Fragmentation ◽  
Induced Apoptosis ◽  
Dynamin 2

Background/Aims: Inhibition of Drp-1-mediated mitochondrial fission limits reactive oxygen species (ROS) production and apoptosis in cardiomyocytes subjected to ischemia/reperfusion injury. It remains unknown if Dynamin 2 inhibition results in similar protective effects. Here we studied the role of Dynamin 2 in cardiomyocyte oxidative stress-induced apoptosis and ROS production. Methods: The effect of lentiviral shRNA (lv5-shRNA) mediated Dynamin 2 knockdown on apopotosis, mitochondria, and ROS production were studied in neonatal mouse cardiomycytes, which were further treated with either selective Drp1 inhibitor mdivi-1 or the Dynamin 2/Drp1 inhibitor Dynasore. Apoptosis was evaluated by flow cytometry. Mitochondrial morphology and transmembrane potential (ΔΨm) were studied by confocal microscopy, and ROS production was detected by dichlorofluorescein diacetate. Results: Inhibition of Drp1 and Dynamin 2 protected against mitochondrial fragmentation, maintained ΔΨm, attenuated cellular ROS production and limited apoptosis. Moreover, Lv5-shRNA mediated knockdown of Dynamin 2 alleviated mitochondrial fragmentation, and reduced both ROS production and oxidative stress-induced apoptosis. The protective effects of Dynamin 2 knockdown were enhanced by Dynasore, indicating an added benefit. Conclusions: Oxidative stress-induced apoptosis and ROS production are attenuated by not only Drp1 inhibition but also Dynamin 2 inhibition, implicating Dynamin 2 as a mediator of oxidative stress in cardiomyocytes.

scholarly journals Cyclin-dependent kinases regulate splice-specific targeting of dynamin-related protein 1 to microtubules

2013 ◽  
Vol 201 (7) ◽  
pp. 1037-1051 ◽  
Author(s):  
Stefan Strack ◽  
Theodore J. Wilson ◽  
J. Thomas Cribbs
Keyword(s):  
Alternative Splicing ◽  
Splice Variants ◽  
Mitochondrial Fission ◽  
Alternative Exon ◽  
Mitochondrial Morphology ◽  
Related Protein ◽  
Fusion Reactions ◽  
Mitochondrial Fragmentation ◽  
Cyclin Dependent Kinases ◽  
And Function

Fission and fusion reactions determine mitochondrial morphology and function. Dynamin-related protein 1 (Drp1) is a guanosine triphosphate–hydrolyzing mechanoenzyme important for mitochondrial fission and programmed cell death. Drp1 is subject to alternative splicing of three exons with previously unknown functional significance. Here, we report that splice variants including the third but excluding the second alternative exon (x01) localized to and copurified with microtubule bundles as dynamic polymers that resemble fission complexes on mitochondria. A major isoform in immune cells, Drp1-x01 required oligomeric assembly and Arg residues in alternative exon 3 for microtubule targeting. Drp1-x01 stabilized and bundled microtubules and attenuated staurosporine-induced mitochondrial fragmentation and apoptosis. Phosphorylation of a conserved Ser residue adjacent to the microtubule-binding exon released Drp1-x01 from microtubules and promoted mitochondrial fragmentation in a splice form–specific manner. Phosphorylation by Cdk1 contributed to dissociation of Drp1-x01 from mitotic microtubules, whereas Cdk5-mediated phosphorylation modulated Drp1-x01 targeting to interphase microtubules. Thus, alternative splicing generates a latent, cytoskeletal pool of Drp1 that is selectively mobilized by cyclin-dependent kinase signaling.

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