TBK1-medicated DRP1 phosphorylation orchestrates mitochondrial dynamics and autophagy activation in osteoarthritis.

Mitochondrial dynamics, including mitochondrial fission and fusion, are critical for maintaining mitochondrial functions. Evidence shows that TANK-binding kinase 1 (TBK1) regulates mitochondrial fusion and fission and then mitophagy. Since a previous study demonstrates a strong correlation between mitophagy and osteoarthritis (OA), we herein investigated the potential role of TBK1 in OA process and mitochondrial functions. We demonstrated a strong correlation between TBK1 and OA, evidenced by significantly downregulated expression of TBK1 in cartilage tissue samples of OA patients and in the chondrocytes of aged mice, as well as TNF-α-stimulated phosphorylation of TBK1 in primary mouse chondrocytes. TBK1 overexpression significantly attenuated TNF-α-induced apoptosis and abnormal mitochondrial function in primary mouse chondrocytes. Furthermore, TBK1 overexpression induced remodeling of mitochondrial morphology by directly phosphorylating dynamin-related protein 1 (DRP1) at Ser637, abolishing the fission of DRP1 and preventing its fragmentation function. Moreover, TBK1 recruitment and DRP1 phosphorylation at Ser637 was necessary for engulfing damaged mitochondria by autophagosomal membranes during mitophagy. Moreover, we demonstrated that APMK/ULK1 signaling contributed to TBK1 activation. In OA mouse models established by surgical destabilization of the medial meniscus, intraarticular injection of lentivirus-TBK1 significantly ameliorated cartilage degradation via regulation of autophagy and alleviation of cell apoptosis. In conclusion, our results suggest that the TBK1/DRP1 pathway is involved in OA and pharmacological targeting of the TBK1-DRP1 cascade provides prospective therapeutic benefits for the treatment of OA.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) as a novel therapeutic target for diseases associated with mitochondrial dysfunction.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a mitochondrial outer membrane-anchored protein-containing transmembrane domain in its N- and C-terminal regions, where both are exposed to the cytosol. Interestingly the C-terminal region has a RING finger domain responsible for its E3 ligase activity, as ubiquitin or in SUMOylation, interacting with proteins related to mitochondrial fusion and fission, cell survival, and tumor suppressor process, such as Akt. Therefore, MUL1 is involved in various cellular processes, such as mitochondrial dynamics, inter-organelle communication, proliferation, mitophagy, immune response, inflammation and cell apoptosis. MUL1 is expressed at a higher basal level in the heart, immune system organs, and blood. Here, we discuss the role of MUL1 in mitochondrial dynamics and its function in various pathological models, both in vitro and in vivo. In this context, we describe the role of MUL1 in: (1) the inflammatory response, by regulating NF-κB activity; (2) cancer, by promoting cell death and regulating exonuclear function of proteins, such as p53; (3) neurological diseases, by maintaining communication with other organelles and interacting with proteins to eliminate damaged organelles and; (4) cardiovascular diseases, by maintaining mitochondrial fusion/fission homeostasis. In this review, we summarize the latest advances in the physiological and pathological functions of MUL1. We also describe the different substrates of MUL1, acting as a positive or negative regulator in various pathologies associated with mitochondrial dysfunction. In conclusion, MUL1 could be a potential key target for the development of therapies that focus on ensuring the functionality of the mitochondrial network and, furthermore, the quality control of intracellular components by synchronously modulating the activity of different cellular mechanisms involved in the aforementioned pathologies. This, in turn, will guide the development of targeted therapies.

Metastatic TNBC is closely associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism.

Mitochondria modify their function and morphology to satisfy the bioenergetic demand of the cells. Cancer cells take advantage of these features to sustain their metabolic, proliferative, metastatic, and survival necessities. Understanding the morphological changes to mitochondria in the different grades of triple-negative breast cancer (TNBC) could help to design new treatments. Consequently, this research explored mitochondrial morphology and the gene expression of some proteins related to mitochondrial dynamics, as well as proteins associated with oxidative and non-oxidative metabolism in metastatic and non-metastatic TNBC. We found that mitochondrial morphology and metabolism are different in metastatic and non-metastatic TNBC. In metastatic TNBC, there is overexpression of genes related to mitochondrial dynamics, fatty-acid metabolism, and glycolysis. These features are accompanied by a fused mitochondrial morphology. By comparison, in non-metastatic TNBC, there is a stress-associated mitochondrial morphology with hyperfragmented mitochondria, accompanied by the upregulated expression of genes associated with the biogenesis of mitochondria; both of which are characteristics related to the higher production of reactive oxygen species observed in this cell line. These differences between metastatic and non-metastatic TNBC should provide a better understanding of metastasis and contribute to the development of improved specific and personalized therapies for TNBC.

Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology.

Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that establishes latency in ganglionic neurons throughout the neuraxis after primary infection. Here, we show that VZV infection induces a time-dependent significant change in mitochondrial morphology, an important indicator of cellular health, since mitochondria are involved in essential cellular functions. VZV immediate-early protein 63 (IE63) was detected in mitochondria-rich cellular fractions extracted from infected human fetal lung fibroblasts (HFL) by Western blotting. IE63 interacted with cytochrome c oxidase in bacterial 2-hybrid analyses. Confocal microscopy of VZV-infected HFL cells at multiple times after infection revealed the presence of IE63 in the nucleus, mitochondria, and cytoplasm. Our data provide the first evidence that VZV infection induces alterations in mitochondrial morphology, including fragmentation, which may be involved in cellular damage and/or death during virus infection.

Get1p and Get2p are required for maintenance of mitochondrial morphology and normal cardiolipin levels.

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. CL deficiency leads to defects in mitochondrial function. Using a targeted synthetic lethality screen to identify defects that exacerbate CL deficiency, we determined that deletion of mitochondrial morphology genes in cells lacking CL leads to severe growth defects. We show that ER membrane proteins Get1p and Get2p are required for maintaining normal levels of CL. We propose that these proteins regulate the level of CL by maintaining wild type-like tubular mitochondrial morphology. The genetic interactions observed in this study identify novel physiological modifiers that are required for maintenance of CL levels and mitochondrial morphology.

Hederagenin suppresses ovarian cancer via targeting mitochondrial fission through dynamin-related protein 1.

A triterpenoid isolated from the plant Hedera helix, hederagenin was discovered to have anti-cancer, anti-inflammatory, anti-depressant and anti-fibrosis properties both in vivo and in vitro. In this study, the relationship between mitochondrial fission and hederagenin-induced apoptosis in ovarian cancer (OC) was investigated and the underlying mechanisms were deciphered. Hederagenin's cytotoxicity on OC cells was analyzed using colony formation and CCK-8 assays. The effect of hederagenin on OC cells was also verified by a mouse xenograft tumor model. Flow cytometric analysis was conducted to examine hederagenin's effects on mitochondrial membrane potential, apoptosis, and cell cycle OC cells. MitoTracker Red (CMXRos) staining was performed to observe the mitochondrial morphology. The protein levels of Bak, Bcl-2, Caspase 3, Caspase 9, Cyclin D1 and Bax were measured by Western blot. This study found that hederagenin could suppress the in vivo and in vitro SKOV3 and A2780 cell proliferation in an effective manner. Besides, hederagenin altered the mitochondrial membrane potential, induced S-phase and G0/G1-phase arrest, mitochondrial morphology changes, and apoptosis in OC cells. Additionally, our findings further demonstrated that hederagenin changed the mitochondrial morphology by suppressing dynamin-related protein 1 (Drp1), a crucial mitochondrial division factor. Moreover, Drp1 overexpression could reverse hederagenin-induced apoptosis, whereas the Drp1 knockdown had the opposite effect. Furthermore, hederagenin may trigger BAX mitochondrial translocation and apoptosis in OC cells. These results provided a novel perspective on the relationship between the modulation of mitochondrial morphology and the suppression of ovarian cancer by hederagenin.

Megamitochondria plasticity: Function transition from adaption to disease.

As the cell's energy factory and metabolic hub, mitochondria are critical for ATP synthesis to maintain cellular function. Mitochondria are highly dynamic organelles that continuously undergo fusion and fission to alter their size, shape, and position, with mitochondrial fusion and fission being interdependent to maintain the balance of mitochondrial morphological changes. However, in response to metabolic and functional damage, mitochondria can grow in size, resulting in a form of abnormal mitochondrial morphology known as megamitochondria. Megamitochondria are characterized by their considerably larger size, pale matrix, and marginal cristae structure and have been observed in various human diseases. In energy-intensive cells like hepatocytes or cardiomyocytes, the pathological process can lead to the growth of megamitochondria, which can further cause metabolic disorders, cell damage and aggravates the progression of the disease. Nonetheless, megamitochondria can also form in response to short-term environmental stimulation as a compensatory mechanism to support cell survival. However, extended stimulation can reverse the benefits of megamitochondria leading to adverse effects. In this review, we will focus on the findings of the different roles of megamitochondria, and their link to disease development to identify promising clinical therapeutic targets.

Morphological fluctuations of individual mitochondria in living cells.

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 livingXenopus laevismelanophores, 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 2.1µm 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.

Determining the Intracellular Organization of Organelles.

Many studies have found alterations in the positioning and morphology of intracellular organelles under different experimental conditions. Although the precise quantification of these changes is challenging, it is strongly facilitated in single cells that are seeded on micropatterned substrates. Indeed, the controlled microenvironment of the cell leads to a reproducible distribution of organelles, simplifying image analysis and minimizing the number of cells required for robust phenotypes. Here, we outline how alterations in the intracellular organization of lysosomes and mitochondria, as a result of different growth conditions, can be efficiently quantified in cells seeded on adhesive micropatterns.

Automatic Mitochondria Segmentation for EM Data Using a 3D Supervised Convolutional Network.

Recent studies have supported the relation between mitochondrial functions and degenerative disorders related to ageing, such as Alzheimer's and Parkinson's diseases. Since these studies have exposed the need for detailed and high-resolution analysis of physical alterations in mitochondria, it is necessary to be able to perform segmentation and 3D reconstruction of mitochondria. However, due to the variety of mitochondrial structures, automated mitochondria segmentation and reconstruction in electron microscopy (EM) images have proven to be a difficult and challenging task. This paper puts forward an effective and automated pipeline based on deep learning to realize mitochondria segmentation in different EM images. The proposed pipeline consists of three parts: (1) utilizing image registration and histogram equalization as image pre-processing steps to maintain the consistency of the dataset; (2) proposing an effective approach for 3D mitochondria segmentation based on a volumetric, residual convolutional and deeply supervised network; and (3) employing a 3D connection method to obtain the relationship of mitochondria and displaying the 3D reconstruction results. To our knowledge, we are the first researchers to utilize a 3D fully residual convolutional network with a deeply supervised strategy to improve the accuracy of mitochondria segmentation. The experimental results on anisotropic and isotropic EM volumes demonstrate the effectiveness of our method, and the Jaccard index of our segmentation (91.8% in anisotropy, 90.0% in isotropy) and F1 score of detection (92.2% in anisotropy, 90.9% in isotropy) suggest that our approach achieved state-of-the-art results. Our fully automated pipeline contributes to the development of neuroscience by providing neurologists with a rapid approach for obtaining rich mitochondria statistics and helping them elucidate the mechanism and function of mitochondria.