Structural basis for recruitment of mitochondrial fission complexes by Fis1

Mitochondrial fission controls mitochondrial shape and physiology, including mitochondrial remodeling in apoptosis. During assembly of the yeast mitochondrial fission complex, the outer membrane protein Fis1 recruits the dynamin-related GTPase Dnm1 to mitochondria. Fis1 contains a tetratricopeptide repeat (TPR) domain and interacts with Dnm1 via the molecular adaptors Mdv1 and Caf4. By using crystallographic analysis of adaptor-Fis1 complexes, we show that these adaptors use two helices to bind to both the concave and convex surfaces of the Fis1 TPR domain. Fis1 therefore contains two interaction interfaces, a binding mode that, to our knowledge, has not been observed previously for TPR domains. Genetic and biochemical studies indicate that both binding interfaces are important for binding of Mdv1 and Caf4 to Fis1 and for mitochondrial fission activity in vivo. Our results reveal how Fis1 recruits the mitochondrial fission complex and will facilitate efforts to manipulate mitochondrial fission.     

ARC5, a cytosolic dynamin-like protein from plants,is part of the chloroplast division machinery

Chloroplast division in plant cells is orchestrated by a complex macromolecular machine with components positioned on both the inner and outer envelope surfaces. The only plastid division proteins identified to date are of endosymbiotic origin and are localized inside the organelle. Employing positional cloning methods in Arabidopsis in conjunction with a novel strategy for pinpointing the mutant locus, we have identified a gene encoding a new chloroplast division protein, ARC5. Mutants of ARC5 exhibit defects in chloroplast constriction, have enlarged, dumbbell-shaped chloroplasts, and are rescued by a wild-type copy of ARC5. The ARC5 gene product shares similarity with the dynamin family of GTPases, which mediate endocytosis, mitochondrial division, and other organellar fission and fusion events in eukaryotes. Phylogenetic analysis showed that ARC5 is related to a group of dynamin-like proteins unique to plants. A GFP-ARC5 fusion protein localizes to a ring at the chloroplast division site. Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast. Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified. ARC5 has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants. These results indicate that the chloroplast division apparatus is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes.     

Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death

A number of pathogenic bacteria target mitochondria to modulate the host's apoptotic machinery. Studies here revealed that infection with the human gastric pathogen Helicobacter pylori disrupts the morphological dynamics of mitochondria as a mechanism to induce host cell death. The vacuolating cytotoxin A (VacA) is both essential and sufficient for inducing mitochondrial network fragmentation through the mitochondrial recruitment and activation of dynamin-related protein 1 (Drp1), which is a critical regulator of mitochondrial fission within cells. Inhibition of Drp1-induced mitochondrial fission within VacA-intoxicated cells inhibited the activation of the proapoptotic Bcl-2-associated X (Bax) protein, permeabilization of the mitochondrial outer membrane, and cell death. Our data reveal a heretofore unrecognized strategy by which a pathogenic microbe engages the host's apoptotic machinery.     

The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission

The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11β and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.Peroxisomes are ubiquitous, single-membrane–bounded cell organelles that harbor enzymes involved in a large number of metabolic processes. Common functions are the β-oxidation of fatty acids and hydrogen peroxide metabolism. Specialized functions include the metabolism of various carbon and organic nitrogen sources in fungi and the production of plasmalogens and bile acids in mammals, to name but a few (1). Their importance is underlined by the severe, often lethal human disorders caused by defects in peroxisome biogenesis or metabolism (2). Importantly, defects in peroxisome multiplication, caused by mutations in genes that control peroxisome fission, also result in severe human disorders (3, 4).Based on data from yeast and mammals, the current model for peroxisomal fission describes a three-step process, consisting of (i) organelle elongation, (ii) constriction, and (iii) the actual scission step (5–7). So far, Peroxin 11 (Pex11p), a highly conserved and abundant peroxisomal membrane protein, is the only protein known to play a crucial role in the first step (8). Its vital role in peroxisome multiplication is illustrated by the observation that in all organisms studied so far, Pex11p overproduction results in enhanced peroxisome proliferation, whereas PEX11 deletion causes a decrease in number, together with an increase in peroxisome size (8). The function of Pex11p in organelle elongation is mediated by the extreme N-terminal region of Pex11p, which can adopt the structure of an amphipathic helix, which upon insertion into membranes induces their curvature, resulting in organelle tubulation (9).The molecular mechanisms of peroxisome constriction are poorly understood. In contrast, several proteins required for the final stage of the fission process are known. The first protein shown to be involved in this process was Saccharomyces cerevisiae Vps1p, a dynamin-like protein (DLP) (10). Later studies revealed that in this organism the DLP Dynamin-related 1 (Dnm1p) is also involved in peroxisome fission, especially under peroxisome-inducing growth conditions (11). Dnm1p forms a fission machinery together with the tail-anchored fission protein Fis1p and (in S. cerevisiae) the accessory proteins Mdv1p and Caf4p (12). Interestingly these proteins are also responsible for mitochondrial fission in yeast (13).Dnm1p (Drp1 in mammals) (11, 14) is a large GTPase that achieves membrane fission by forming oligomeric, ring-like structures around constricted sites on organelle membranes (15). Powered by GTP hydrolysis, these ring-like structures then tighten further until the membrane severs. Interestingly, Dnm1p is recruited to Pex11p-enriched elongated peroxisomal membranes, suggesting that Pex11p and Dnm1p are functionally linked (16, 17).Here, we identify a previously unknown role for Pex11p in peroxisomal fission. We show that Pex11p directly interacts with Dnm1p and that this interaction stimulates the GTPase activity of Dnm1p, establishing Pex11p as a GTPase activating protein (GAP) that plays a crucial role in the last step of the peroxisome fission process.     

Single-membrane–bounded peroxisome division revealed by isolation of dynamin-based machinery

Peroxisomes (microbodies) are ubiquitous single-membrane–bounded organelles and fulfill essential roles in the cellular metabolism. They are found in virtually all eukaryotic cells and basically multiply by division. However, the mechanochemical machinery involved in peroxisome division remains elusive. Here, we first identified the peroxisome-dividing (POD) machinery. We isolated the POD machinery from Cyanidioschyzon merolae, a unicellular red alga containing a single peroxisome. Peroxisomal division in C. merolae can be highly synchronized by light/dark cycles and the microtubule-disrupting agent oryzalin. By proteomic analysis based on the complete genome sequence of C. merolae, we identified a dynamin-related protein 3 (DRP3) ortholog, CmDnm1 (Dnm1), that predominantly accumulated with catalase in the dividing-peroxisome fraction. Immunofluorescence microscopy demonstrated that Dnm1 formed a ring at the division site of the peroxisome. The outlines of the isolated dynamin rings were dimly observed by phase-contrast microscopy and clearly stained for Dnm1. Electron microscopy revealed that the POD machinery was formed at the cytoplasmic side of the equator. Immunoelectron microscopy showed that the POD machinery consisted of an outer dynamin-based ring and an inner filamentous ring. Down-regulation of Dnm1 impaired peroxisomal division. Surprisingly, the same Dnm1 serially controlled peroxisomal division after mitochondrial division. Because genetic deficiencies of Dnm1 orthologs in multiperoxisomal organisms inhibited both mitochondrial and peroxisomal proliferation, it is thought that peroxisomal division by contraction of a dynamin-based machinery is universal among eukaryotes. These findings are useful for understanding the fundamental systems in eukaryotic cells.Peroxisomes are single-membrane–bounded organelles found in nearly all eukaryotic cells. In plant cells, peroxisomes are involved in a variety of metabolic pathways essential for development associated with photorespiration, lipid mobilization, and hormone biosynthesis (1, 2). In animals, abnormalities in peroxisome proliferation are associated with carcinogenesis, neurodegeneration, and cerebrohepatorenal syndrome (1, 3). Peroxisomes are thought to basically proliferate by division, although they do not contain DNA (1). Because the cells of multiperoxisomal organisms, such as yeasts, plants, and animals, contain irregularly shaped peroxisomes that divide randomly, their proliferation has been examined by analyzing peroxisome abundance and distribution (4, 5). Therefore, the division machinery (ring) that is essential for proliferation and plays a central role is unclear. Cyanidioschyzon merolae offers unique advantages for studying peroxisomal division, because each cell contains a minimal set of basic eukaryotic organelles, comprising one chloroplast, one mitochondrion, one cell nucleus, and one peroxisome, the divisions of which occur in that order and can be synchronized by light/dark cycles (6–9) (Fig. 1 A and B and Fig. S1). In C. merolae, peroxisomes do not form de novo from the endoplasmic reticulum in the peroxisomal division cycle but divide by binary fission (6, 7, 10). In addition, the complete sequence of the genome has enabled proteomic analyses (7, 11).Open in a separate windowFig. 1.Identification of Dnm1 from the dividing-peroxisome fraction. (A) Immunofluorescence and schematic images of mitochondrial and peroxisomal divisions of C. merolae. Peroxisomal (red) division occurred after mitochondrial (yellow) division. Chl, chloroplast; Mt, mitochondrion; Nu, nucleus; PC, phase-contrast image; Po, peroxisome. (B) Frequencies of dividing cell nuclei (Nu), dividing chloroplasts (Chl), dividing mitochondria (Mt), and dividing peroxisomes (Po) in non–oryzalin-treated cells (control) and oryzalin-treated cells (Orz+) at the indicated times after synchronization (n > 100). (C) Proteomic analysis of peroxisomal fractions in control and oryzalin-treated cells at 20 h after synchronization. The major bands specific to oryzalin-treated cells were identified as catalase (black arrowhead), Dnm1 (red arrowhead), and others. (D) Immunoblot analyses of Dnm1, catalase, mitochondria division protein (Mda1), porin, and chloroplast division protein (PDR1). Cell, whole cell; Mt/Chl, isolated mitochondria and chloroplast; Po, isolated peroxisomes. (Scale bars: 1 μm.)     

A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division

Recently, the FtsZ protein, which is known as a key component in bacterial cell division, was reported to be involved in mitochondrial division in algae. In yeast and animals, however, mitochondrial fission depends on the dynamin-like proteins Dnm1p and Drp1, respectively, whereas in green plants, no potential mitochondrial division genes have been identified. BLAST searches of the nuclear and mitochondrial genome sequences of Arabidopsis thaliana did not find any obvious homologue of the alpha-proteobacterial-type ftsZ genes. To determine whether mitochondrial division of higher plants depends on a dynamin-like protein, we cloned a cDNA for ADL2b, an Arabidopsis homologue of Dnm1p, and tested its subcellular localization and its dominant-negative effect on mitochondrial division. The fusion protein of green fluorescent protein and ADL2b was observed as punctate structures localized at the tips and at the constriction sites of mitochondria in live plant cells. Cells expressing dominant-negative mutant ADL2b proteins (K56A and T77F) showed a significant fusion, aggregation, and/or tubulation of mitochondria. We propose that mitochondrial division in higher plants is conducted by dynamin-like proteins similar to ADL2b in Arabidopsis. The evolutional points of loss of mitochondrial FtsZ and the functional acquisition of dynamin-like proteins in mitochondrial division are discussed.     

OPA1 requires mitofusin 1 to promote mitochondrial fusion

The regulated equilibrium between mitochondrial fusion and fission is essential to maintain integrity of the organelle. Mechanisms of mitochondrial fusion are largely uncharacterized in mammalian cells. It is unclear whether OPA1, a dynamin-related protein of the inner membrane mutated in autosomal dominant optic atrophy, participates in fusion or fission. OPA1 promoted the formation of a branched network of elongated mitochondria, requiring the integrity of both its GTPase and C-terminal coiled-coil domain. Stable reduction of OPA1 levels by RNA interference resulted in small, fragmented, and scattered mitochondria. Levels of OPA1 did not affect mitochondrial docking, but they correlated with the extent of fusion as measured by polyethylene glycol mitochondrial fusion assays. A genetic analysis proved that OPA1 was unable to tubulate and fuse mitochondria lacking the outer membrane mitofusin 1 but not mitofusin 2. Our data show that OPA1 functionally requires mitofusin 1 to regulate mitochondrial fusion and reveal a specific functional difference between mitofusin 1 and 2.     

动力相关蛋白1抑制剂减轻糖尿病小鼠心肌缺血/再灌注损伤

目的 探究动力相关蛋白（Dynamin-related protein,Drp）1抑制剂Mdivi-1对糖尿病小鼠心肌缺血/再灌注（Myocardial ischemia/reperfusion,MI/R）损伤的作用及其机制。方法 高脂饮食加小剂量链脲佐菌素（STZ）诱导建立糖尿病小鼠模型。造模成功的糖尿病小鼠进行MI/R处理,再灌注前15 min腹腔注射Mdivi-1(1.2 mg/kg)或其溶剂二甲基亚砜。主要评价指标包括线粒体形态、心脏功能、心肌损伤及凋亡,蛋白免疫印迹检测Drp1表达。结果 糖尿病MI/R后线粒体分裂增加（P<0.01）,线粒体Drp1转位明显增加（P<0.01）,Mdivi-1可抑制缺血/再灌注心肌的Drp1线粒体转位及线粒体分裂,减少心肌梗死面积和心肌细胞凋亡（P<0.01）,减轻氧化应激（P<0.05）。结论 Drp1介导的线粒体分裂增加参与了糖尿病MI/R损伤,Drp1抑制剂Mdivi-1可抑制线粒体分裂,减轻糖尿病MI/R损伤。     

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.     

线粒体分裂对胰岛β细胞功能的影响

目的 探讨线粒体分裂对胰岛β细胞功能的影响.方法 通过可诱导表达野生型动力相关蛋白1(Drp-1 WT)基因和突变型Drp-1(Drp-1K38A)基因的大鼠INS-1 β细胞,分析不同葡萄糖条件下,线粒体分裂对胰岛β细胞功能的影响.结果 在高糖条件下,强力霉素诱导后的Drp-1WT细胞线粒体分裂过程增强,网络状结构部分断裂,多呈点状结构,细胞胰岛素分泌功能降低(P<0.01),线粒体膜电位降低(P<0.05),细胞内ATP含量减少(P<0.05),细胞色素C表达增加,而在Drp-1K38A细胞中,上述变化明显减轻.结论 线粒体分裂增强可抑制胰岛β细胞功能.

Abstract：

Objective To investigate the effect of mitochondrial fission on the function of pancreatic β cells.Methods INS-1 stable cell lines allowing inducible expression of either wild-type dynamin-related protein 1 (Drp-1 WT)or its dominant-negative mutant(Drp-1 K38A)were used.The effect of mitochondrial fission on the function of pancreatic β cells were investigated under different concentrations of glucose.Results There were increased mitochondrial fission and disintegration of the mitochondrial reticulum into multiple punctiform organelles in Drp-1 WT cells induced with doxycycline under high glucose condition.Insulin secretion(P<0.01),mitochondrial membrane potential(P<0.05),and ATP content(P<0.05)were decreased and cytochrome C expression was increased after the expression of Drp-1 WT under high glucose condition while these changes were markedly mild in Drp-1 K38A expression cells.Conclusion The increased mitochondrial fission inhibits pancreatic β cell function.     

Pharmacological activation of AMPK prevents Drp1-mediated mitochondrial fission and alleviates endoplasmic reticulum stress-associated endothelial dysfunction

Background and purposeThis study aims to investigate whether and how pharmacological activation of AMP-activated protein kinase (AMPK) improves endothelial function by suppressing mitochondrial ROS-associated endoplasmic reticulum stress (ER stress) in the endothelium.Experimental approachPalmitate stimulation induced mitochondrial fission and ER stress-associated endothelial dysfunction. The effects of AMPK activators salicylate and AICA riboside (AICAR) on mitochondrial ROS production, Drp1 phosphorylation, mitochondrial fission, ER stress, thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation, inflammation, cell apoptosis and endothelium-dependent vasodilation were observed.Key results“Silencing” of TXNIP by RNA interference inhibited NLRP3 inflammasome activation in response to ER stress, indicating that TXNIP was a key link between ER stress and NLRP3 inflammasome activation. AMPK activators salicylate and AICAR prevented ROS-induced mitochondrial fission by enhancing dynamin-related protein 1 (Drp1) phosphorylation (Ser 637) and thereby attenuated IRE-1α and PERK phosphorylation, but their actions were blocked by knockdown of AMPK. Salicylate and AICAR reduced TXNIP induction and inhibited NLRP3 inflammasome activation by reducing NLRP3 and caspase-1 expression, leading to a reduction in IL-1β secretion. As a result, salicylate and AICAR inhibited inflammation and reduced cell apoptosis. Meanwhile, salicylate and AICAR enhanced eNOS phosphorylation and restored the loss of endothelium-dependent vasodilation in the rat aorta. Immunohistochemistry staining showed that AMPK activation inhibited ER stress and NLRP3 inflammasome activation in the vascular endothelium.Conclusion and implicationsPharmacological activation of AMPK regulated mitochondrial morphology and ameliorated endothelial dysfunction by suppression of mitochondrial ROS-associated ER stress and subsequent TXNIP/NLRP3 inflammasome activation. These findings suggested that regulation of Drp1 phosphorylation by AMPK activation contributed to suppression of ER stress and thus presented a potential therapeutic strategy for AMPK activation in the regulation of endothelium homeostasis.     

An ancestral bacterial division system is widespread in eukaryotic mitochondria

Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such as Saccharomyces cerevisiae, mammals, and Arabidopsis thaliana seem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid Andalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.     

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.     

Dynamic recruitment of dynamin for final mitochondrial severance in a primitive red alga

Dynamins are a eukaryote-specific family of GTPases. Some family members are involved in diverse and varied cellular activities. Here, we report that the primitive red alga Cyanidioschyzon merolae retains only one dynamin homolog, CmDnm1, belonging to the mitochondrial division subfamily. Previously, the bacterial cell division protein, FtsZ, was shown to localize at the mitochondrial division site in the alga. We showed that FtsZ and dynamin coexist as mitochondrial division-associated proteins that act during different phases of division. CmDnm1 was recruited from 10-20 cytoplasmic patches (dynamin patches) to the midpoint of the constricted mitochondrion-dividing ring (MD ring), which was observed as an electron-dense structure on the cytoplasmic side. CmDnm1 is probably not required for early constriction; it forms a ring or spiral when the outer mitochondrial membrane is finally severed, whereas the FtsZ and MD rings are formed before constriction. It is thought that the FtsZ, MD, and dynamin rings are involved in scaffolding, constriction, and final separation, respectively. In eukaryotes, mitochondrial severance is probably the most conserved role for the dynamin family.     

PTEN-induced kinase 1-induced dynamin-related protein 1 Ser637 phosphorylation reduces mitochondrial fission and protects against intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R) occurs in various diseases, such as trauma and intestinal transplantation. Excessive reactive oxygen species(ROS)accumulation and subsequent apoptotic cell death in intestinal epithelia are important causes of I/R injury. PTEN-induced putative kinase 1(PINK1) and phosphorylation of dynamin-related protein 1(DRP1) are critical regulators of ROS and apoptosis. However, the correlation of PINK1 and DRP1 and their function in intestinal I/R injury have not been investigated. Thus, examining the PINK1/DRP1 pathway may help to identify a protective strategy and improve the patient prognosis.AIM To clarify the mechanism of the PINK1/DRP1 pathway in intestinal I/R injury.METHODS Male C57 BL/6 mice were used to generate an intestinal I/R model via superior mesenteric artery occlusion followed by reperfusion. Chiu's score was used to evaluate intestinal mucosa damage. The mitochondrial fission inhibitor mdivi-1 was administered by intraperitoneal injection. Caco-2 cells were incubated in vitro in hypoxia/reoxygenation conditions. Small interfering RNAs and overexpression plasmids were transfected to regulate PINK1 expression. The protein expression levels of PINK1, DRP1, p-DRP1 and cleaved caspase 3 were measured by Western blotting. Cell viability was evaluated using a Cell Counting Kit-8 assay and cell apoptosis was analyzed by TUNEL staining. Mitochondrial fission and ROS were tested by Mito Tracker and Mito SOX respectively.RESULTS Intestinal I/R and Caco-2 cell hypoxia/reoxygenation decreased the expression of PINK1 and p-DRP1 Ser637. Pretreatment with mdivi-1 inhibited mitochondrial fission, ROS generation, and apoptosis and ameliorated cell injury in intestinal I/R. Upon PINK1 knockdown or overexpression in vitro, we found that p-DRP1 Ser637 expression and DRP1 recruitment to the mitochondria were associated with PINK1. Furthermore, we verified the physical combination of PINK1 and pDRP1 Ser637.CONCLUSION PINK1 is correlated with mitochondrial fission and apoptosis by regulating DRP1 phosphorylation in intestinal I/R. These results suggest that the PINK1/DRP1 pathway is involved in intestinal I/R injury, and provide a new approach for prevention and treatment.     

Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-x_L protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-x_L is abundant. Here, we report that overexpression of Bcl-x_L postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-x_L, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-x_L or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-x_L appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-x_L coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-x_L protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-x_L positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.     

Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release

The ESCRT machinery functions in topologically equivalent membrane fission events, namely multivesicular body formation, the terminal stages of cytokinesis and HIV-1 release. Here, we show that the ESCRT-III-binding protein Alix is recruited to the midbody of dividing cells through binding Cep55 via an evolutionarily conserved peptide. Disruption of Cep55/Alix/ESCRT-III interactions causes formation of aberrant midbodies and cytokinetic failure, demonstrating an essential role for these proteins in midbody morphology and cell division. We also show that the C terminus of Alix encodes a multimerization activity that is essential for its function in Alix-dependent HIV-1 release and for interaction with Tsg101. Last, we demonstrate that overexpression of Chmp4b and Chmp4c differentially inhibits HIV-1 release and cytokinesis, suggesting possible reasons for gene expansion within the mammalian Class E VPS pathway.     

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.     

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.     

Melatonin prevents Drp1‐mediated mitochondrial fission in diabetic hearts through SIRT1‐PGC1α pathway

Myocardial contractile dysfunction is associated with an increase in mitochondrial fission in patients with diabetes. However, whether mitochondrial fission directly promotes diabetes‐induced cardiac dysfunction is still unknown. Melatonin exerts a substantial influence on the regulation of mitochondrial fission/fusion. This study investigated whether melatonin protects against diabetes‐induced cardiac dysfunction via regulation of mitochondrial fission/fusion and explored its underlying mechanisms. Here, we show that melatonin prevented diabetes‐induced cardiac dysfunction by inhibiting dynamin‐related protein 1 (Drp1)‐mediated mitochondrial fission. Melatonin treatment decreased Drp1 expression, inhibited mitochondrial fragmentation, suppressed oxidative stress, reduced cardiomyocyte apoptosis, improved mitochondrial function and cardiac function in streptozotocin (STZ )‐induced diabetic mice, but not in SIRT 1^?/? diabetic mice. In high glucose‐exposed H9c2 cells, melatonin treatment increased the expression of SIRT 1 and PGC ‐1α and inhibited Drp1‐mediated mitochondrial fission and mitochondria‐derived superoxide production. In contrast, SIRT 1 or PGC ‐1α siRNA knockdown blunted the inhibitory effects of melatonin on Drp1 expression and mitochondrial fission. These data indicated that melatonin exerted its cardioprotective effects by reducing Drp1‐mediated mitochondrial fission in a SIRT 1/PGC ‐1α‐dependent manner. Moreover, chromatin immunoprecipitation analysis revealed that PGC ‐1α directly regulated the expression of Drp1 by binding to its promoter. Inhibition of mitochondrial fission with Drp1 inhibitor mdivi‐1 suppressed oxidative stress, alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. These findings show that melatonin attenuates the development of diabetes‐induced cardiac dysfunction by preventing mitochondrial fission through SIRT 1‐PGC 1α pathway, which negatively regulates the expression of Drp1 directly. Inhibition of mitochondrial fission may be a potential target for delaying cardiac complications in patients with diabetes.