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mitoKeima detection of Gp78-dependent basal mitophagy in HT-1080 cells. Wild-type HT-1080 cells and g2-41 knockout CRISPR clones were transfected with mito-Keima and treated with DMSO or CCCP for 24 h. Confocal fluorescence images show mito-Keima in both neutral (yellow) and low pH (red) environments. Accumulation of mitolysosomes (red) is observed selectively in HT-1080 cells and increased upon CCCP treatment. Quantification of both the number of mitolysosomes per cell and the total area of mitolysosomes per cell is shown in the bar graphs (Scale bar: 10 μm; n = 3 independent biological replicates; > 10 cells/condition per experiment; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; Mean ± SEM)
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Mitochondria are major sources of cytotoxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, that when uncontrolled contribute to cancer progression. Maintaining a finely tuned, healthy mitochondrial population is essential for cellular homeostasis and survival. Mitophagy, the selective elimination of mitochondria by autopha...
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Citations
... Thus, the fraction of motile mitochondria following CCCP did not exceed 4-5% in any direction. Nevertheless, it should be noted that different cell types and mitochondria may respond differently to CCCP, and the direction and magnitude of effect on mitochondrial voltage may depend on the age, cell type, timing, and concentration of the drug [32,33]. ...
Presenilin 1 (PS1) is a transmembrane proteolytic subunit of γ-secretase that cleaves amy-loid precursor proteins. Mutations in PS1 (mPS1) are associated with early-onset familial Alzheimer's disease (AD). The link between mutated PS1, mitochondrial calcium regulation, and AD has been studied extensively in different test systems. Despite the wide-ranging role of mPS1 in AD, there is a paucity of information on the link between PS1 and neuronal cell death, a hallmark of AD. In the present study, we employed the selective mitochondrial uncoupler carbonyl cyanide chloro-phenylhydrazone (CCCP) and compared the reactivity of mPS1-transfected cultured rat hippocam-pal neurons with PS1 and control neurons in a situation of impaired mitochondrial functions. CCCP causes a slow rise in cytosolic and mitochondrial calcium in all three groups of neurons, with the mPS1 neurons demonstrating a faster rise. Consequently, mPS1 neurons were depolarized by CCCP and measured with TMRM, a mitochondrial voltage indicator, more than the other two groups. Morphologically, CCCP produced more filopodia in mPS1 neurons than in the other two groups, which were similarly affected by the drug. Finally, mPS1 transfected neurons tended to die from prolonged exposure to CCCP sooner than the other groups, indicating an increase in vulnerability associated with a lower ability to regulate excess cytosolic calcium.
... Notably, the ROS level induced by 30 ug/mL toltrazuril was not significantly different from the H2O2 positive control ( Figure 5A). Mitochondria is a major source of intracellular ROS in most cell types [28], so we used MitoSOX Red (MitoSOX) to detect mitochondrial ROS. The results showed that the pattern of mitochondrial ROS production was almost like that of intracellular ROS ( Figure 5B). ...
... Notably, the ROS level induced by 30 µg/mL toltrazuril was not significantly different from the H 2 O 2 positive control ( Figure 5A). Mitochondria is a major source of intracellular ROS in most cell types [28], so we used MitoSOX Red (MitoSOX) to detect mitochondrial ROS. The results showed that the pattern of mitochondrial ROS production was almost like that of intracellular ROS ( Figure 5B). ...
Intestinal coccidiosis is a common parasitic disease in livestock, caused by the infection of Eimeria and Cystoisospora parasites, which results in great economic losses to animal husbandry. Triazine compounds, such as toltrazuril and diclazuril, are widely used in the treatment and chemoprophylaxis of coccidiosis. Unfortunately, widespread drug resistance has compromised their effectiveness. Most studies have focused on prophylaxis and therapeutics with toltrazuril in flocks, while a comprehensive understanding of how toltrazuril treatment alters the transcriptome of E. tenella remains unknown. In this study, merozoites of E. tenella were treated in vitro with 0.5 μg/mL toltrazuril for 0, 1, 2 and 4 h, respectively. The gene transcription profiles were then compared by high-throughput sequencing. Our results showed that protein hydrolysis genes were significantly upregulated after drug treatment, while cell cycle-related genes were significantly downregulated, suggesting that toltrazuril may affect parasite division. The expression of redox-related genes was upregulated and elevated levels of ROS and autophagosomes were detected in the parasite after toltrazuril treatment, suggesting that toltrazuril may cause oxidative stress to parasite cells and lead to its autophagy. Our results provide basic knowledge of the response of Eimeria genes to toltrazuril and further analysis of the identified transcriptional changes can provide useful information for a better understanding of the mechanism of action of toltrazuril against Eimeria.
... It has been experimentally demonstrated that elevated ROS not only exacerbates damage to the mitochondria themselves [39] , but is also a trigger for ERS. Furthermore, prolonged and severe ERS can lead to the induction of apoptosis in stressed cells [40] . ...
Acute myeloid leukemia (AML) is a disease of malignant proliferation of abnormally or poorly differentiated myeloid cells of the hematopoietic system. The clinical treatments of non-M3 AML are experiencing a lack of effective drugs. V8 is a newly synthesized derivative of the natural flavonoid wogonin, which belongs to the potential anticancer drug, and has shown significant antitumor activity in vitro and in vivo . In this study, we investigated the effects of V8 on AML cell lines and primary AML cells and the underlying mechanisms. The results showed that V8 inhibited the growth and induced the apoptosis of AML cell lines (ME-1, Kasumi-1, and U937) and primary AML cells in a concentration-dependent manner. Meanwhile, we revealed that V8-induced apoptosis was accompanied by mitochondrial injury, such as the reduction of mitochondrial membrane potential and ATP production, activation of endoplasmic reticulum stress (ERS) characterized by GRP78 and caspase-12 expression upregulation. Mechanism studies have shown that V8 induced mitochondrial injury and inhibited mitophagy via elevating the intracellular ROS level. In addition, V8 activated PERK-p-eIF2α-ATF4 and Ire1α-XBP1 pathways and induced apoptosis of AML cells via selectively activating CHOP. Correspondingly, the degree of apoptosis and expression of apoptosis-related proteins were alleviated after the elimination of cytoplasmic ROS with N-Acetyl-L-cysteine (NAC) and knocking out CHOP in the cells by transfection with CHOP shRNA, implicating that mitochondrial injury-triggered upregulation of ROS and CHOP played an important role in V8-induced apoptosis of AML cells. In primary AML cells-bearing NOD/SCID mice model and U937 cells-inoculating BALB/c nude mice xenografts transplantation tumor model, administration of V8 markedly prolonged survival time and inhibited the xenografts growth via CHOP-mediated ERS in vivo . In conclusion, our study provides a new insight into the mechanism of V8-induced apoptosis, suggesting the potential of V8 as a promising agent against AML.
... Using CRISPR/Cas knockout Gp78 HT-1080 cells that present deficient basal mitophagy, impaired mitochondrial health and increased mitochondrial ROS (Alan et al., 2022), we show that loss of Gp78 reduces MERC mitochondrial coverage (Fig. 4A). Surface coverage and number of contacts per mitochondrial surface region are significantly reduced relative to HT-1080 cells but remain elevated compared to COS-7 cells (Fig. 4B). ...
... Morphologically distinct riboMERCs are wider, include ribosomes in the intervening space and are abundantly expressed in liver (Anastasia et al., 2021;Csordas et al., 2006). They are less abundant in most cultured cells, as shown here in our analysis of COS-7 cells, but were found to be abundant by EM analysis of HT-1080 fibrosarcoma cells, that express robust amounts of the Gp78 ubiquitin ligase (Alan et al., 2022;Tsai et al., 2007). Gp78 knockdown in HT-1080 cells selectively reduced riboMERC expression (Wang et al., 2015), a result we confirm here using CRISPR/Cas Gp78 KO HT-1080 cells. ...
... As for tubular matrix ER and ER sheets (Nixon-Abell et al., 2016;Sun et al., 2020), the functional and morphological relationship between tubular riboMERCs and sheet-like WrappER remains to be determined. WrappER is implicated in lipid homeostasis and recently shown to interact with peroxisomes in liver (Anastasia et al., 2021;Ilacqua et al., 2022) while Gp78 ubiquitin ligase activity in HT-1080 cells mediates basal mitophagy, promoting mitochondrial health and reducing mitochondrial ROS (Alan et al., 2022). Further definition of these and, potentially, other functions for riboMERCs requires further study. ...
Identification and morphological analysis of mitochondria-ER contacts (MERCs) by fluorescent microscopy is limited by sub-pixel resolution inter-organelle distances. Application of a Membrane Contact Site (MCS) detection algorithm, MCS-DETECT, to 3D STED super-resolution image volumes reconstructs sub-resolution MERCs. MCS-DETECT shows that elongated ribosome-studded riboMERCs, present in HT-1080 but not COS-7 cells, are morphologically distinct from smaller smooth contacts and larger contacts induced by mitochondria-ER linker expression in COS-7 cells. riboMERC expression is reduced in Gp78 knockout HT-1080 cells and induced by Gp78 ubiquitin ligase activity in COS-7 cells. Knockdown of the riboMERC tether RRBP1 eliminates riboMERCs in both wild-type and Gp78 knockout HT-1080 cells. By MCS-DETECT, Gp78-dependent riboMERCs present complex tubular shapes that intercalate between and contact multiple mitochondria, that are lost upon RRBP1 knockdown. MCS-DETECT of 3D whole cell super-resolution image volumes therefore identifies a novel dual regulatory mechanism for tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.
ETOC Summary
Application of the sub-pixel resolution Membrane Contact Site (MCS) detection algorithm, MCS-DETECT, to 3D STED super-resolution image volumes identifies a novel dual regulatory mechanism for tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.
Various assaults on mitochondria occur during the human aging process, contributing to mitochondrial dysfunction. This mitochondrial dysfunction is intricately connected with aging and diseases associated with it. In vivo, the accumulation of defective mitochondria can precipitate inflammatory and oxidative stress, thereby accelerating aging. Mitophagy, an essential selective autophagy process, plays a crucial role in managing mitochondrial quality control and homeostasis. It is a highly specialized mechanism that systematically removes damaged or impaired mitochondria from cells, ensuring their optimal functioning and survival. By engaging in mitophagy, cells are able to maintain a balanced and stable environment, free from the potentially harmful effects of dysfunctional mitochondria. An ever-growing body of research highlights the significance of mitophagy in both aging and age-related diseases. Nonetheless, the association between mitophagy and inflammation or oxidative stress induced by mitochondrial dysfunction remains ambiguous. We review the fundamental mechanisms of mitophagy in this paper, delve into its relationship with age-related stress, and propose suggestions for future research directions.
Caveolin‐1 (CAV1), the main structural component of caveolae, is phosphorylated at tyrosine‐14 (pCAV1), regulates signal transduction, mechanotransduction, and mitochondrial function, and plays contrasting roles in cancer progression. We report that CRISPR/Cas9 knockout (KO) of CAV1 increases mitochondrial oxidative phosphorylation, increases mitochondrial potential, and reduces ROS in MDA‐MB‐231 triple‐negative breast cancer cells. Supporting a role for pCAV1, these effects are reversed upon expression of CAV1 phosphomimetic CAV1 Y14D but not non‐phosphorylatable CAV1 Y14F. pCAV1 is a known effector of Rho‐associated kinase (ROCK) signaling and ROCK1/2 signaling mediates CAV1 promotion of increased mitochondrial potential and decreased ROS production in MDA‐MB‐231 cells. CAV1/ROCK control of mitochondrial potential and ROS is caveolae‐independent as similar results were observed in PC3 prostate cancer cells lacking caveolae. Increased mitochondrial health and reduced ROS in CAV1 KO MDA‐MB‐231 cells were reversed by knockdown of the autophagy protein ATG5, mitophagy regulator PINK1 or the mitochondrial fission protein Drp1 and therefore due to mitophagy. Use of the mitoKeima mitophagy probe confirmed that CAV1 signaling through ROCK inhibited basal mitophagic flux. Activation of AMPK, a major mitochondrial homeostasis protein inhibited by ROCK, is inhibited by CAV1‐ROCK signaling and mediates the increased mitochondrial potential, decreased ROS, and decreased basal mitophagy flux observed in wild‐type MDA‐MB‐231 cells. CAV1 regulation of mitochondrial health and ROS in cancer cells therefore occurs via ROCK‐dependent inhibition of AMPK. This study therefore links pCAV1 signaling activity at the plasma membrane with its regulation of mitochondrial activity and cancer cell metabolism through control of mitophagy.
Identification and morphological analysis of mitochondria–ER contacts (MERCs) by fluorescent microscopy is limited by subpixel resolution interorganelle distances. Here, the membrane contact site (MCS) detection algorithm, MCS-DETECT, reconstructs subpixel resolution MERCs from 3D super-resolution image volumes. MCS-DETECT shows that elongated ribosome-studded riboMERCs, present in HT-1080 but not COS-7 cells, are morphologically distinct from smaller smooth contacts and larger contacts induced by mitochondria–ER linker expression in COS-7 cells. RiboMERC formation is associated with increased mitochondrial potential, reduced in Gp78 knockout HT-1080 cells and induced by Gp78 ubiquitin ligase activity in COS-7 and HeLa cells. Knockdown of riboMERC tether RRBP1 eliminates riboMERCs in both wild-type and Gp78 knockout HT-1080 cells. By MCS-DETECT, Gp78-dependent riboMERCs present complex tubular shapes that intercalate between and contact multiple mitochondria. MCS-DETECT of 3D whole-cell super-resolution image volumes, therefore, identifies novel dual control of tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.
Endoplasmic reticulum-associated protein degradation (ERAD) is a stringent quality control mechanism through which misfolded, unassembled and some native proteins are targeted for degradation to maintain appropriate cellular and organelle homeostasis. Several in vitro and in vivo ERAD-related studies have provided mechanistic insights into ERAD pathway activation and its consequent events; however, a majority of these have investigated the effect of ERAD substrates and their consequent diseases affecting the degradation process. In this review, we present all reported human single-gene disorders caused by genetic variation in genes that encode ERAD components rather than their substrates. Additionally, after extensive literature survey, we present various genetically manipulated higher cellular and mammalian animal models that lack specific components involved in various stages of the ERAD pathway.
