Mitochondrial dynamics in heart failure

Mitochondria have been widely studied for their critical role in cellular metabolism, energy production, and cell death. New developments in research on mitochondria derived from studies in yeast have led to the discovery of entirely new mitochondrial processes that have implications for mitochondrial function in heart failure. Recent studies have identified that maintaining normal mitochondrial morphology and function depends on the dynamic balance of mitochondrial fusion and fission (division). Mitochondrial fusion and fission are constant ongoing processes, which are essential for the maintenance of normal mitochondrial function. Studies in heart failure have been limited but suggest a possible reduction in mitochondrial fusion. As mitochondrial fusion and fission have important links to apoptosis, a key mechanism of loss of cardiac myocytes in heart failure, there are many implications for both heart failure research and treatment.     

Mitochondria in heart failure: the emerging role of mitochondrial dynamics

Over the past decade, mitochondria have emerged as critical integrators of energy production, generation of reactive oxygen species (ROS), multiple cell death, and signaling pathways in the constantly beating heart. Clarification of the molecular mechanisms, underlying mitochondrial ROS generation and ROS-induced cell death pathways, associated with cardiovascular diseases, by itself remains an important aim; more recently, mitochondrial dynamics has emerged as an important active mechanism to maintain normal mitochondria number and morphology, both are necessary to preserve cardiomyocytes integrity. The two opposing processes, division (fission) and fusion, determine the cell type-specific mitochondrial morphology, the intracellular distribution and activity. The tightly controlled balance between fusion and fission is of particular importance in the high energy demanding cells, such as cardiomyocytes, skeletal muscles, and neuronal cells. A shift toward fission will lead to mitochondrial fragmentation, observed in quiescent cells, while a shift toward fusion will result in the formation of large mitochondrial networks, found in metabolically active cardiomyocytes. Defects in mitochondrial dynamics have been associated with various human disorders, including heart failure, ischemia reperfusion injury, diabetes, and aging. Despite significant progress in our understanding of the molecular mechanisms of mitochondrial function in the heart, further focused research is needed to translate this knowledge into the development of new therapies for various ailments.     

Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery

Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinson's disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. Here, we show that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. Our genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention.     

Mitochondrial dynamics and cell death in heart failure

The highly regulated processes of mitochondrial fusion (joining), fission (division) and trafficking, collectively called mitochondrial dynamics, determine cell-type specific morphology, intracellular distribution and activity of these critical organelles. Mitochondria are critical for cardiac function, while their structural and functional abnormalities contribute to several common cardiovascular diseases, including heart failure (HF). The tightly balanced mitochondrial fusion and fission determine number, morphology and activity of these multifunctional organelles. Although the intracellular architecture of mature cardiomyocytes greatly restricts mitochondrial dynamics, this process occurs in the adult human heart. Fusion and fission modulate multiple mitochondrial functions, ranging from energy and reactive oxygen species production to Ca²⁺ homeostasis and cell death, allowing the heart to respond properly to body demands. Tightly controlled balance between fusion and fission is of utmost importance in the high energy-demanding cardiomyocytes. A shift toward fission leads to mitochondrial fragmentation, while a shift toward fusion results in the formation of enlarged mitochondria and in the fusion of damaged mitochondria with healthy organelles. Mfn1, Mfn2 and OPA1 constitute the core machinery promoting mitochondrial fusion, whereas Drp1, Fis1, Mff and MiD49/51 are the core components of fission machinery. Growing evidence suggests that fusion/fission factors in adult cardiomyocytes play essential noncanonical roles in cardiac development, Ca²⁺ signaling, mitochondrial quality control and cell death. Impairment of this complex circuit causes cardiomyocyte dysfunction and death contributing to heart injury culminating in HF. Pharmacological targeting of components of this intricate network may be a novel therapeutic modality for HF treatment.     

Molecular mechanisms mediating mitochondrial dynamics and mitophagy and their functional roles in the cardiovascular system

Mitochondria are essential organelles that produce the cellular energy source, ATP. Dysfunctional mitochondria are involved in the pathophysiology of heart disease, which is associated with reduced levels of ATP and excessive production of reactive oxygen species. Mitochondria are dynamic organelles that change their morphology through fission and fusion in order to maintain their function. Fusion connects neighboring depolarized mitochondria and mixes their contents to maintain membrane potential. In contrast, fission segregates damaged mitochondria from intact ones, where the damaged part of mitochondria is subjected to mitophagy whereas the intact part to fusion. It is generally believed that mitochondrial fusion is beneficial for the heart, especially under stress conditions, because it consolidates the mitochondria's ability to supply energy. However, both excessive fusion and insufficient fission disrupt the mitochondrial quality control mechanism and potentiate cell death. In this review, we discuss the role of mitochondrial dynamics and mitophagy in the heart and the cardiomyocytes therein, with a focus on their roles in cardiovascular disease. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".     

烟酰胺核糖抑制1型糖尿病大鼠心肌线粒体分裂的作用及机制

目的 探讨烟酰胺核糖（nicotinamide riboside, NR）对1型糖尿病（DM）大鼠心肌线粒体融合分裂的作用及其机制。 方法 利用链脲佐菌素（streptozotocin, STZ）诱导1型DM大鼠模型,随机分为4组:正常对照（Con）组,Con+NR组,DM组,DM+NR组。监测大鼠血糖和体质量变化,葡萄糖耐量试验（IPGTT）检测糖代谢情况,采用小动物超声评估大鼠心脏功能变化,TUNEL法检测心肌细胞凋亡,DHE染色检测心肌细胞ROS含量,透射电镜观察线粒体形态,Western blot法检测线粒体分裂、融合相关蛋白表达水平。 结果 与Con组相比,DM组大鼠的血糖、血脂明显升高,体质量减轻,心脏左室射血分数（left ventricular ejection fraction, LVEF）和左心室短轴缩短率（Left ventricular fractional shortening, LVFS）减低,心肌细胞凋亡增加,氧化应激增强,线粒体分裂增多,线粒体融合蛋白Opa1表达下调（P<0.05, P<0.01）;与DM组相比,DM+NR组大鼠心脏LVEF改善,心肌细胞凋亡减少,氧化应激减少,线粒体融合增加,线粒体融合蛋白Opa1和Mfn2表达上调（P<0.05）。 结论 NR可增加DM心肌融合蛋白Opa1与Mfn2的表达,抑制DM心肌线粒体分裂,降低DM心肌凋亡和氧化应激,改善心脏功能。     

Mitochondrial-Shaping Proteins in Cardiac Health and Disease – the Long and the Short of It!

Mitochondrial health is critically dependent on the ability of mitochondria to undergo changes in mitochondrial morphology, a process which is regulated by mitochondrial shaping proteins. Mitochondria undergo fission to generate fragmented discrete organelles, a process which is mediated by the mitochondrial fission proteins (Drp1, hFIS1, Mff and MiD49/51), and is required for cell division, and to remove damaged mitochondria by mitophagy. Mitochondria undergo fusion to form elongated interconnected networks, a process which is orchestrated by the mitochondrial fusion proteins (Mfn1, Mfn2 and OPA1), and which enables the replenishment of damaged mitochondrial DNA. In the adult heart, mitochondria are relatively static, are constrained in their movement, and are characteristically arranged into 3 distinct subpopulations based on their locality and function (subsarcolemmal, myofibrillar, and perinuclear). Although the mitochondria are arranged differently, emerging data supports a role for the mitochondrial shaping proteins in cardiac health and disease. Interestingly, in the adult heart, it appears that the pleiotropic effects of the mitochondrial fusion proteins, Mfn2 (endoplasmic reticulum-tethering, mitophagy) and OPA1 (cristae remodeling, regulation of apoptosis, and energy production) may play more important roles than their pro-fusion effects. In this review article, we provide an overview of the mitochondrial fusion and fission proteins in the adult heart, and highlight their roles as novel therapeutic targets for treating cardiac disease.     

Mitochondrial energetics and insulin resistance

Obesity, insulin resistance, type 2 diabetes mellitus, and aging are associated with impaired skeletal muscle oxidation capacity, reduced mitochondrial content, and lower rates of oxidative phosphorylation. Several studies have reported ultrastructural abnormalities in mitochondrial morphology and reductions in mitochondrial mass in insulin-resistant individuals. From lower organisms to rodents, mitochondrial membrane structure, function, and programmed cell death are regulated in part by the balance between the opposing forces of mitochondrial fusion and fission, suggesting they may also play an important role in human physiology.     

The PINK1/Parkin pathway regulates mitochondrial morphology

Loss-of-function mutations in the PTEN-induced kinase 1 (PINK1) or parkin genes, which encode a mitochondrially localized serine/threonine kinase and a ubiquitin-protein ligase, respectively, result in recessive familial forms of Parkinsonism. Genetic studies in Drosophila indicate that PINK1 acts upstream of Parkin in a common pathway that influences mitochondrial integrity in a subset of tissues, including flight muscle and dopaminergic neurons. The mechanism by which PINK1 and Parkin influence mitochondrial integrity is currently unknown, although mutations in the PINK1 and parkin genes result in enlarged or swollen mitochondria, suggesting a possible regulatory role for the PINK1/Parkin pathway in mitochondrial morphology. To address this hypothesis, we examined the influence of genetic alterations affecting the machinery that governs mitochondrial morphology on the PINK1 and parkin mutant phenotypes. We report that heterozygous loss-of-function mutations of drp1, which encodes a key mitochondrial fission-promoting component, are largely lethal in a PINK1 or parkin mutant background. Conversely, the flight muscle degeneration and mitochondrial morphological alterations that result from mutations in PINK1 and parkin are strongly suppressed by increased drp1 gene dosage and by heterozygous loss-of-function mutations affecting the mitochondrial fusion-promoting factors OPA1 and Mfn2. Finally, we find that an eye phenotype associated with increased PINK1/Parkin pathway activity is suppressed by perturbations that reduce mitochondrial fission and enhanced by perturbations that reduce mitochondrial fusion. Our studies suggest that the PINK1/Parkin pathway promotes mitochondrial fission and that the loss of mitochondrial and tissue integrity in PINK1 and parkin mutants derives from reduced mitochondrial fission.     

Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology

Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the molecular mechanism behind mitochondrial morphology has been recruited to govern novel functions, such as development, calcium signaling, and apoptosis, which suggests that novel mechanisms should exist to regulate the conserved membrane fusion/fission machinery. Here we show that phosphorylation and cleavage of the vertebrate-specific Pbeta domain of the mammalian presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology. Phosphorylation of three residues embedded in this domain, Ser-65, Thr-69, and Ser-70, impair a cleavage at position Ser(77)-Ala(78) that is required to initiate PARL-induced mitochondrial fragmentation. Our findings reveal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology.     

Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology

Increased production of mitochondrial reactive oxygen species (ROS) by hyperglycemia is recognized as a major cause of the clinical complications associated with diabetes and obesity [Brownlee, M. (2001) Nature 414, 813-820]. We observed that dynamic changes in mitochondrial morphology are associated with high glucose-induced overproduction of ROS. Mitochondria undergo rapid fragmentation with a concomitant increase in ROS formation after exposure to high glucose concentrations. Neither ROS increase nor mitochondrial fragmentation was observed after incubation of cells with the nonmetabolizable stereoisomer L-glucose. However, inhibition of mitochondrial pyruvate uptake that blocked ROS increase did not prevent mitochondrial fragmentation in high glucose conditions. Importantly, we found that mitochondrial fragmentation mediated by the fission process is a necessary component for high glucose-induced respiration increase and ROS overproduction. Extended exposure to high glucose conditions, which may mimic untreated diabetic conditions, provoked a periodic and prolonged increase in ROS production concomitant with mitochondrial morphology change. Inhibition of mitochondrial fission prevented periodic fluctuation of ROS production during high glucose exposure. These results indicate that the dynamic change of mitochondrial morphology in high glucose conditions contributes to ROS overproduction and that mitochondrial fission/fusion machinery can be a previously unrecognized target to control acute and chronic production of ROS in hyperglycemia-associated disorders.     

Diabetes regulates mitochondrial biogenesis and fission in mouse neurons

Aims/hypothesis  

Normal mitochondrial activity is a critical component of neuronal metabolism and function. Disruption of mitochondrial activity by altered mitochondrial fission and fusion is the root cause of both neurodegenerative disorders and Charcot–Marie–Tooth type 2A inherited neuropathy. This study addressed the role of mitochondrial fission in the pathogenesis of diabetic neuropathy.     

The systems biology of mitochondrial fission and fusion and implications for disease and aging

Mitochondria organize themselves as dynamic populations within a cell, by undergoing continuous cycles of fission and fusion. The spatio-temporal distribution and abundance of mitochondria determines the cell’s energy budget and is thus intimately linked to the cell’s response to environmental stimuli during aging. The dynamic balance of mitochondrial fission and fusion can be studied in terms of antagonistic subpopulations that regulate the mitochondrial responses in space and time. The dynamic nature of these processes motivates mathematical modelling and the simulation of such complex process. In several neurodegenerative and metabolic diseases the dynamic balance of fission and fusion is disturbed. However, how this dynamics plays a role in the progression of diseases is largely unclear. Fission and fusion help mitochondria to regulate cellular energy (ATP) levels, and minimize accumulation of harmful oxidized material called reactive oxygen species which accelerate mutations in mitochondrial DNA (mtDNA) during aging. We discuss how systems biology approaches can be used to investigate the mechanisms controlling the fission–fusion dynamics under two categories: dissecting the design of its molecular regulatory motifs, and understanding complex mitochondrial responses through their population level interactions. This will help us to understand how different regulatory mechanisms regulate the ATP and mutation (mtDNA) landscape of mitochondria to a variety of environmental stimuli in order to maintain their function during aging.     

A molecular switch that governs mitochondrial fusion and fission mediated by the BCL2-like protein CED-9 of Caenorhabditis elegans

Depending on the cellular context, BCL2-like proteins promote mitochondrial fusion or fission. What determines which of these two opposing processes they promote has so far been unknown. Furthermore, the mechanisms through which BCL2-like proteins affect mitochondrial dynamics remain to be fully understood. The BCL2-like protein CED-9 of Caenorhabditis elegans has previously been shown to promote mitochondrial fusion by physically interacting with the mitochondrial fusion protein FZO-1. Here, we report that CED-9 also physically interacts with the mitochondrial fission protein DRP-1 and that this interaction can be enhanced when CED-9 is associated with the BH3-only protein EGL-1. In addition, we show that the EGL-1-CED-9 complex promotes mitochondrial fission by recruiting DRP-1 to mitochondria and that the egl-1 gene is required for CED-9-dependent mitochondrial fission in vivo. Based on these results, we propose that EGL-1 converts CED-9 into a mitochondrial receptor for DRP-1, thereby shifting its activity from profusion to profission. We hypothesize that BCL2-like proteins act as mitochondrial receptors for DRP-1-like proteins in higher organisms as well and that BH3-only proteins play a general role as modifiers of the function in mitochondrial dynamics of BCL2-like proteins. We speculate that this function of BCL2-like proteins may be as couplers of mitochondrial fusion and fission.     

Mst1敲除减轻高脂饮食诱发的心肌线粒体动力学紊乱

目的 探讨哺乳动物STE20样激酶1（Mst1）基因敲除是否可以减轻高脂饮食导致的心肌线粒体动力学紊乱并探究其机制。 方法 C57BL/6小鼠（WT）和Mst1基因敲除小鼠（Mst1-/-）(C57BL/6背景)随机分为正常饮食（Normal diet,ND）组和高脂饮食（High-fat diet,HFD）组,连续喂养16w后,检测各组小鼠体质量、心脏重量/胫骨长度和空腹血脂水平,超声心动图评估心脏功能,HE染色及透射电镜观察心肌肥厚和线粒体形态,心肌柠檬酸合酶(CS)活性和ATP含量评价线粒体功能,Western blot检测线粒体动力学相关蛋白的表达水平。 结果 与ND+WT组相比,HFD+WT组小鼠出现肥胖、高血脂、心肌肥厚和心脏舒张功能障碍,同时线粒体分裂增多、形态明显受损,心肌CS活性、ATP含量均降低,线粒体分裂蛋白Drp1、Fis1的表达明显上调（P<0.05）,融合蛋白Mfn2的表达明显下调（P<0.05）;与HFD+WT组相比,HFD+Mst1-/-组小鼠心脏舒张功能、线粒体形态与功能的损伤明显减轻,Drp1表达明显下调（P<0.05）,Mfn2表达明显上调（P<0.05）。 结论 Mst1基因敲除可以通过抑制线粒体分裂,促进线粒体融合,以维持线粒体动力学稳定,从而减轻脂毒性相关心肌损害。     

Morphological dynamics of mitochondria — A special emphasis on cardiac muscle cells

Mitochondria play a critical role in cellular energy metabolism, Ca²⁺ homeostasis, reactive oxygen species generation, apoptosis, aging, and development. Many recent publications have shown that a continuous balance of fusion and fission of these organelles is important in maintaining their proper function. Therefore, there is a steep correlation between the form and function of mitochondria. Many major proteins involved in mitochondrial fusion and fission have been identified in different cell types, including heart. However, the functional role of mitochondrial dynamics in the heart remains, for the most part, unexplored. In this review we will cover the recent field of mitochondrial dynamics and its physiological and pathological implications, with a particular emphasis on the experimental and theoretical basis of mitochondrial dynamics in the heart.     

Melatonin prevents the dynamin‐related protein 1‐dependent mitochondrial fission and oxidative insult in the cortical neurons after 1‐methyl‐4‐phenylpyridinium treatment

Mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of Parkinson's disease (PD). Mitochondrial morphology is dynamic and precisely regulated by the mitochondrial fission and fusion machinery. Aberrant mitochondrial fragmentation controlled by the mitochondrial fission protein, dynamin‐related protein 1 (Drp1), may result in cell death. Our previous results showed that melatonin protected neurons by inhibiting oxidative stress in a 1‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced PD model. However, the effect of melatonin on mitochondrial dynamics remains uncharacterized. Herein, we investigated the effect of melatonin and the role of Drp1 on MPP⁺‐induced mitochondrial fission in rat primary cortical neurons. We found that MPP⁺ induced a rapid increase in the ratio of GSSG:total glutathione (a marker of oxidative stress) and mitochondrial fragmentation, Drp1 upregulation within 4 hours, and finally resulted in neuron loss 48 hours after the treatment. Neurons overexpressing wild‐type Drp1 promoted mitochondrial and nuclear fragmentation; however, neurons overexpressing dominant‐negative Drp1^K38A or cotreated with melatonin exhibited significantly reduced MPP⁺‐induced mitochondrial fragmentation and neuron death. Moreover, melatonin cotreatment prevented an MPP⁺‐induced high ratio of GSSG and mitochondrial Drp1 upregulation. The prevention of mitochondrial fission by melatonin was not found in neurons transfected with wild‐type Drp1. These results provide a new insight that the neuroprotective effect of melatonin against MPP⁺ toxicity is mediated by inhibiting the oxidative stress and Drp1‐mediated mitochondrial fragmentation.     

Integrity of the yeast mitochondrial genome,but not its distribution and inheritance,relies on mitochondrial fission and fusion

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin–dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.Mitochondrial DNA (mtDNA) is essential for respiratory growth of all eukaryotic cells, and all multicellular organisms depend on mtDNA for their development. Not surprisingly, given the fundamental importance of mtDNA, mutations within mtDNA have been identified as the cause for a plethora of human diseases (1). mtDNA in Saccharomyces cerevisiae encodes for seven essential subunits of the respiratory chain, one protein and two RNA subunits of the mitochondrial ribosome, 24 tRNAs, and the RNA subunit of RNase P (2). Every cell contains 50–100 copies of mtDNA that are organized into nucleoprotein complexes termed nucleoids, each containing 1–10 copies of mtDNA (3, 4). Nucleoids are distributed throughout the mitochondrial network, which is likely important for equivalently supplying spatially separated mitochondrial segments with mitochondrially encoded proteins.How the distribution of mtDNA throughout the mitochondrial network is established and maintained is not fully understood. Previous work from our laboratory and others has shown that the movement of nucleoids within the mitochondrial network is limited, suggesting that the mechanisms of nucleoid distribution are tightly interlinked with the dynamics of mitochondria themselves (5, 6). Mitochondria undergo constant fusion and fission events that are mediated by dedicated machineries, with the central components Fzo1 and Dnm1 required for fusion and fission, respectively (7). Recently, we have provided support for a role of mitochondrial fission in mtDNA distribution. We have shown that mtDNA localizes to sites of Dnm1-dependent mitochondrial fission and that it is segregated after scission to both of the newly generated mitochondrial tips (8). Localizing mtDNA to the newly formed tips would then allow transport of mitochondrial tips and mtDNA to distal parts in the cell, where fusion with the mitochondrial network may drive mtDNA distribution. Such a mechanism would be particularly important during inheritance of mtDNA to daughter buds during cell division, where mtDNA needs to be transported over a relatively large distance. In S. cerevisiae, mitochondria are inherited in a myosin- and F-actin–dependent process, in which a mitochondrial tubule invades the budding daughter cell and is subsequently anchored at the distal membrane (9). An active mtDNA partition and inheritance apparatus has been postulated (6); however, the spatiotemporal relationship between the inheritance of mitochondria and the inheritance of mtDNA has not been examined.If mitochondrial fusion and fission were essential for the distribution and inheritance of mtDNA, their loss would impair the process. Indeed, fusion-defective cells lose mtDNA (10, 11), most likely due to excessive fragmentation. By contrast, however, fission-defective cells, as well as cells defective in fusion and fission, remain capable of respiratory growth, indicating that a functional mitochondrial genome must be maintained (10, 12). These observations suggest that fission-independent mechanisms must exist that facilitate mtDNA inheritance.In this work, we investigated the role of mitochondrial fusion and fission in mtDNA distribution and inheritance. Through the development of a noninvasive method to quantify the spatial organization of mtDNA within mitochondrial tubules, we found that cells deficient in fusion and fission maintain a WT distribution of mtDNA. Live-cell imaging showed that this distribution is facilitated by the de novo generation of tubules from the sides of existing tubules, a process coupled to the spatial positioning of mtDNA to the newly formed tip. Unexpectedly, although dispensable for maintaining mtDNA distribution and inheritance, fusion and fission were required to maintain the integrity of the mitochondrial genome.     

Atypical mitochondrial fission upon bacterial infection

We recently showed that infection by Listeria monocytogenes causes mitochondrial network fragmentation through the secreted pore-forming toxin listeriolysin O (LLO). Here, we examine factors involved in canonical fusion and fission. Strikingly, LLO-induced mitochondrial fragmentation does not require the traditional fission machinery, as Drp1 oligomers are absent from fragmented mitochondria following Listeria infection or LLO treatment, as the dynamin-like protein 1 (Drp1) receptor Mff is rapidly degraded, and as fragmentation proceeds efficiently in cells with impaired Drp1 function. LLO does not cause processing of the fusion protein optic atrophy protein 1 (Opa1), despite inducing a decrease in the mitochondrial membrane potential, suggesting a unique Drp1- and Opa1-independent fission mechanism distinct from that triggered by uncouplers or the apoptosis inducer staurosporine. We show that the ER marks LLO-induced mitochondrial fragmentation sites even in the absence of functional Drp1, demonstrating that the ER activity in regulating mitochondrial fission can be induced by exogenous agents and that the ER appears to regulate fission by a mechanism independent of the canonical mitochondrial fission machinery.Mitochondria are essential organelles that perform a multitude of functions, ranging from the production of biosynthetic intermediates and energy to innate immune signaling and cellular calcium buffering or the storage of proapoptotic components (1). To perform these diverse functions, mitochondria respond to cellular cues and display a highly variable and dynamic morphology, constantly undergoing fusion and fission. It is becoming increasingly clear that mitochondrial dynamics and function are deeply interconnected, and mitochondrial dysfunction is associated with a range of diseases.Wild-type mitochondrial morphology and function are maintained by a balance between mitochondrial fusion and fission. Fusion allows exchange of genetic material between single mitochondria and is mediated by two large guanosine triphosphate phosphohydrolases (GTPases) embedded in the outer membrane (mitofusin 1 and 2) and an inner membrane GTPase, Opa1 (2). Deletion mutants affecting these three proteins accumulate dysfunctional mitochondria, leading to neurodegenerative phenotypes and different forms of myopathy (1, 3).Mitochondrial fusion is balanced by fission, which is essential to ensure proper distribution of mitochondria and energy supply to daughter cells in mitosis or within a single cell. This necessity is particularly evident in neurons, where fission defects prevent efficient mitochondrial transport to synapses, the crucial sites of energy consumption (4, 5). The physiological importance of mitochondrial fission is further highlighted by its essential role in embryonic development in mice and nematodes (6–8).Mitochondrial fission is thought to be accomplished by the dynamin-like protein Drp1, a mainly cytosolic protein that is recruited to future fission sites, where it oligomerizes to form spirals that constrict mitochondria. Mitochondrial fission is regulated at several levels: by initial ER- and actin-mediated mitochondrial constriction (9, 10), leading to the accumulation of the membrane-bound Drp1 receptor Mff and by several posttranslational modifications of Drp1, which modulate its activity (11).Listeria monocytogenes is a foodborne pathogen capable of invading nonphagocytic cells, where it can replicate and spread. The pathogenic potential of L. monocytogenes correlates with the expression of several virulence genes (12). One of the most important virulence factors is listeriolysin O (LLO), a highly regulated secreted pore-forming toxin (reviewed in ref. 13). LLO belongs to the family of cholesterol-dependent cytolysins (CDCs), most of which are produced by extracellular bacteria such as Streptococci or Clostridia. CDCs oligomerize on cholesterol-containing membranes to form nonselective ion-permeable pores of variable sizes (14) that act in concert with bacterial phospholipases to allow bacterial escape from the phagosome. More recently, LLO has been found to have several intracellular and extracellular roles that extend beyond phagosomal escape. For example, we have shown that infection with L. monocytogenes causes fragmentation of the host mitochondrial network by action of its pore-forming toxin LLO before bacterial entry (15).In this study, we demonstrate that LLO-induced mitochondrial fragmentation does not follow canonical pathways, because it is independent of key fusion and fission components, such as Opa1 and Drp1. We demonstrate that the ER marks mitochondrial fragmentation sites even in the absence of functional Drp1, and that the actin cytoskeleton also facilitates fragmentation. LLO-induced fragmentation is distinct from that observed upon treatment with uncouplers [such as carbonyl cyanide m-chlorophenylhydrazone (CCCP)] and apoptosis inducers (such as staurosporine), revealing a unique pathway for mitochondrial fragmentation that can be induced by an exogenous agent.