‘Porosome’ discovered nearly 20 years ago provides molecular insights into the kiss‐and‐run mechanism of cell secretion

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950s, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of an additional mechanism that allows partial discharge of intra‐vesicular contents during secretion. This proposed mechanism requires the involvement of a plasma membrane structure called ‘porosome’, which serves to prevent the collapse of secretory vesicles, and to transiently fuse with the plasma membrane (Kiss‐and‐run), expel a portion of its contents and disengage. Porosomes are cup‐shaped supramolecular lipoprotein structures at the cell plasma membrane ranging in size from 15 nm in neurons and astrocytes to 100–180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins. In comparison, the 120 nm nuclear pore complex is composed of >500 protein molecules. Elucidation of the porosome structure, its chemical composition and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex resulting in the establishment of membrane continuity to form a fusion pore at the porosome base, has been demonstrated. Additionally, the molecular mechanism of secretory vesicle swelling, and its requirement for intra‐vesicular content release during cell secretion has also been elucidated. Collectively, these observations provide a molecular understanding of cell secretion, resulting in a paradigm shift in our understanding of the secretory process.     

Stress‐induced OMA1 activation and autocatalytic turnover regulate OPA1‐dependent mitochondrial dynamics

The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin‐like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1‐mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress‐sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1‐mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival.     

The mechanical effects of CRT promoting autophagy via mitochondrial calcium uniporter down‐regulation and mitochondrial dynamics alteration

The mechanism of cardiac resynchronization therapy (CRT) remains unclear. In this study, mitochondria calcium uniporter (MCU), dynamin‐related protein‐1 (DNM1L/Drp1) and their relationship with autophagy in heart failure (HF) and CRT are investigated. Thirteen male beagle's dogs were divided into three groups (sham, HF, CRT). Animals received left bundle branch (LBB) ablation followed by either 8‐week rapid atrial pacing or 4‐week rapid atrial pacing and 4‐week biventricular pacing. Cardiac function was evaluated by echocardiography. Differentially expressed genes (DEGs) were detected by microarray analysis. General morphological changes, mitochondrial ultrastructure, autophagosomes and mitophagosomes were investigated. The cardiomyocyte stretching was adopted to imitate the mechanical effect of CRT. Cells were divided into three groups (control, angiotensin‐II and angiotensin‐II + stretching). MCU, DNM1L/Drp1 and autophagy markers were detected by western blots or immunofluorescence. In the present study, CRT could correct cardiac dysfunction, decrease cardiomyocyte's size, alleviate cardiac fibrosis, promote the formation of autophagosome and mitigate mitochondrial injury. CRT significantly influenced gene expression profile, especially down‐regulating MCU and up‐regulating DNM1L/Drp1. Cell stretching reversed the angiotensin‐II induced changes of MCU and DNM1L/Drp1 and partly restored autophagy. CRT's mechanical effects down‐regulated MCU, up‐regulated DNM1L/Drp1 and subsequently enhanced autophagy. Besides, the mechanical stretching prevented the angiotensin‐II‐induced cellular enlargement.     

‘Not anymore,not yet’: Australian–Hungarians rediscover the ‘old‐new’ Hungary

This article examines the return visits of Australian‐Hungarians to their homeland after 1989 and the different types of homecoming experiences after an extended period of separation. The focus of the paper is returnees’ perceptions of changes to Hungary or lack thereof. I analyse the distinction between the ‘desired/nostalgic past’, which Hungarian returnees nurtured during the decades of separation and expected to rediscover upon return, and the ‘disdained past’ of the Communist dictatorship, which they had fled and hoped never to encounter again. The findings demonstrate that people interpret both past and present in relation to hopes, expectations and disappointments situated within particular nationalist imaginaries, political projects and ideological prisms. This allows us to analyse the nexus between local, national and diasporic belonging through post‐socialist identities, orientations toward democracy and understandings of ‘nation’.     

Mitofusins ‘bridge’ the gap between oxidative stress and mitochondrial hyperfusion

During stress conditions, mitochondria can undergo hyperfusion to protect the cell. A recent study in EMBO reports identifies a new mechanism by which mitofusins can be activated to initiate mitochondrial during oxidative stress.EMBO reports (2012) 13, 909–915; doi:10.1038/embor.2012.128Mitochondria are dynamic organelles that undergo fission and fusion events, but we are only beginning to understand some of the reasons and the machineries involved in these processes [1]. Fission allows distribution of mitochondria to daughter cells following mitosis. Small mitochondria are more efficiently tracked along the cytoskeleton, which also allows a quality control mechanism to exist where fragmented ‘old'' mitochondria can be turned over by autophagy [2]. Fusion enables mitochondrial contents to be mixed between neighbouring organelles. Fission and fusion events must be tightly regulated, and whilst they are opposing processes, they seem to act in concert together—for example, fusion events often lead to a subsequent fission event at the same site. In this issue of EMBO reports, McBride and colleagues [3] identify a new mechanism by which mitofusins can be activated to initiate mitochondrial fusion under conditions of oxidative stress.…GSSG stimulates mitochondrial fusion whereas GSH inhibits itFission involves surface receptors and adaptors (Fis1, Mff, MiD49 and MiD51/MIEF1) and the cytosolic dynamin-related protein Drp1. Fusion involves the dynamin GTPases mitofusin (Mfn) 1 and 2 at the outer and Opa1 at the inner membrane. Mitofusins form homo- and hetero-oligomers to tether adjacent mitochondria together. How the outer membranes fuse before Opa1 acts to fuse the inner membranes is not known. Moreover, the mechanisms regulating their fusion activity are not well-understood. Mitochondrial fission and fusion are important in developmental and physiological processes. Mitochondrial fragmentation occurs during apoptosis and necrosis, where the loss of the network facilitates cell death. However, under conditions of less severe stress, mitochondria can undergo hyperfusion, seemingly as a mechanism by which they can protect the cell from dying [4]. During nutrient starvation, which leads to the induction of macro-autophagy, mitochondria also undergo hyperfusion to prevent their encapsulation by autophagosomes. Hyperfusion is also important during the G1 to S transition of the cell cycle. Until now, the mechanisms regulating stress-induced mitochondrial hyperfusion have remained elusive.McBride and colleagues used an in vitro fusion assay, which consists of mixing mitochondria isolated from cell cultures expressing either an amino-terminal or a carboxy-terminal domain of luciferase, targeted to the mitochondrial matrix. Both luciferase domains contained a leucine zipper that leads to dimerization upon mitochondrial fusion thereby generating a functional enzyme. The authors found that in vitro, mitochondrial fusion was stimulated by oxidative stress including hydrogen peroxide treatment, whereas anti-oxidants inhibited the process. They then tested whether fusion might be responsive to reactive oxygen species or cellular oxidants. Glutathione (GSH), found in all parts of the cell, provides the main redox buffer for cells. GSH contains a free thiol group, and the formation of a disulphide bond between two GSH molecules gives rise to oxidized GSH (GSSG). GSH reductase recycles GSSG to GSH thus maintaining the cellular redox state. During oxidative stress, GSSG accumulates and can interact with other proteins, and induces either glutathionylation or the generation of a disulphide bond known as ‘disulphide switching''. McBride and colleagues found that GSSG stimulates mitochondrial fusion whereas GSH inhibits it. The fusion reaction could be stimulated by adding GSSG directly to isolated mitochondria whilst it was inhibited by the cysteine alkylating agent iodoacetate. This implies that mitochondrial proteins with free thiol groups are direct substrates of GSH-mediated oxidation and that this regulates mitochondrial fusion. Analysis of Mfn1 and Mfn2 revealed that GSSG treatment induces oligomer formation, which could be resolved on non-reducing SDS–PAGE as four distinct species of 160–220 kDa. Addition of a reductant led to the loss of the oligomeric species on SDS–PAGE, suggesting that the mitofusins might indeed form disulphide bonds. The presence of discrete oligomeric species on non-reducing gels suggests that intermolecular disulphides might occur and that the mitofusins interact with additional proteins. Interestingly, two proteins shown to regulate mitofusin activity directly, SLP2 and non-apoptotic Bax, were not modified by oxidant. Similarly, Drp1 was unaffected, pointing to a direct activation of fusion rather than a decrease in fission activity. However, it remains possible that fission might also be downregulated by oxidation, and inactivation, of mitochondrial fission receptors such as Fis1, Mff and MiD49/MiD51 or by a different post-translational modification of Drp1. The authors validated their work by incubating cultured cells with agents that cause an increase in the levels of GSSG. Such treatment induced mitochondrial hyperfusion as expected, but it also resulted in the increase of Mfn2 oligomers.This implies that mitochondrial proteins with free thiol groups are direct substrates of GSH-mediated oxidation and that this regulates mitochondrial fusionMitofusins contain a cytosolically exposed N-terminal GTPase domain followed by a heptad repeat (HR1), and two transmembrane regions that form a hairpin in the mitochondrial outer membrane allowing the exposure of another heptad repeat (HR2) to the cytosol. The heptad repeats are involved in membrane-tethering events, and structural analysis of a HR2 dimer revealed that it forms an antiparallel coiled-coil [5]. When the authors added non-hydrolysable GTP to their in vitro assay, the GSSG-mediated fusion was blocked. When mitochondria were diluted to reduce tethering between mitochondria, GSSG still induced mitofusin oligomerization and this depended on GTP hydrolysis. This suggests that stress-induced oligomerization occurs largely between mitofusin molecules on the surface of the same mitochondria—that is, in cis. At higher concentrations of mitochondria where tethering events are enhanced, GTP hydrolysis did not seem to be required to induce oligomerization of oxidized mitofusins between adjacent mitochondria—that is, in trans. To narrow down the cysteine residues that are oxidized in mitofusins, the authors performed site-directed mutagenesis of Mfn2 and introduced the mutants into Mfn2^−/− mouse embryonic fibroblasts. The authors found that mutation of Cys 684 in the hinge region of HR2 reduced the formation of oligomers and was less functional in rescuing mitochondrial fusion. The authors suggest that disulphide bond formation between this residue and cysteines in adjacent mitofusins might alter the coiled-coil interactions, thereby priming the proteins to activate fusion.…disulphide bond formation between […] adjacent mitofusins might alter the coiled-coil interactions, thereby priming the proteins to activate fusionWhy is hyperfusion needed? Mitochondrial fragmentation is an important aspect of cell death pathways, and hyperfusion might open a window of time to enable the cell to activate proteins involved in responding to cellular insults. However, cells can also block fusion during stress-induced apoptosis. It was found that stress-activated c-Jun N-terminal kinase phosphorylates Mfn2, leading to its ubiquitination and degradation [6]. How cells decide whether to turn on either survival mechanisms that activate mitofusins, or death mechanisms that inactivate them, requires additional studies.The work by McBride and colleagues points to the importance of intracellular redox conditions in regulating mitochondrial fusion. Changes in cellular GSH might also be important for regulating other members of the fission–fusion machinery. Ganglioside-induced differentiation associated protein 1 (GDAP1) functions in mitochondrial fission, and mutations in GDAP1 lead to the neurodegenerative disorder Charcot Marie-Tooth disease (CMT) 2A. GDAP1 contains GSH S-transferase domains, and loss of the protein has been found to decrease the levels of GSH [7]. Interestingly, mutations in Mfn2 lead to CMT4A, which is clinically almost indistinct from CMT2A. A study of a family with CMT revealed an asymptomatic mother with a mutation in GDAP1 and an asymptomatic father with a mutation in Mfn2. Their child who inherited both mutant alleles developed severe neuropathy [8]. The cumulative effects of inheriting both mutations points to an overlap in the function of these proteins. The new findings, pointing to the importance of GSH in mitochondrial fusion, warrant investigation into whether a closer connection between GDAP1 and mitofusins exists.? Open in a separate windowFigure 1Model for mitochondrial fusion induced by oxidative stress. Oxidation of cysteines in mitofusins induces oligomer formation in cis, probably through the formation of one or more disulphide bonds, which might cause a conformational change in the heptad repeat (HR) regions aiding in tethering to mitofusins in trans to enhance membrane fusion.     

‘I am helping them’: ‘Traffickers’, ‘anti‐traffickers’ and economies of bad faith

For several years, aid programs in the Mekong region have taken an increasing interest in cross‐border mobility and human trafficking and its relationship with development. More recently, there has been an increasing interest in the identification of trafficked victims and the investigation, arrest and prosecution of traffickers. Whereas anti‐trafficking programs ubiquitously define themselves as being in a battle with traffickers, this article argues that although they are not homologous social actors, both engage in acts of bad faith. The article elaborates this argument by drawing attention to the recruitment process within the Lao sex industry as well as to the way in which aid programs attempt to identify trafficked victims. It concludes that imaginary aspects of development underpin a simultaneous disjuncture yet enable the social reproduction of the life worlds of ‘traffickers’ and ‘anti‐traffickers’ alike.     

Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition

F‐ATP synthases convert the electrochemical energy of the H⁺ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca²⁺‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca²⁺ binding site and the mechanism(s) through which Ca²⁺ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca²⁺‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca²⁺‐ATP hydrolysis, confer resistance to Ca²⁺‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.     

Acetyl‐l‐carnitine protects yeast cells from apoptosis and aging and inhibits mitochondrial fission

In this work we report that carnitines, in particular acetyl‐l‐ carnitine (ALC), are able to prolong the chronological aging of yeast cells during the stationary phase. Lifespan extension is significantly reduced in yca1 mutants as well in rho⁰ strains, suggesting that the protective effects pass through the Yca1 caspase and mitochondrial functions. ALC can also prevent apoptosis in pro‐apoptotic mutants, pointing to the importance of mitochondrial functions in regulating yeast apoptosis and aging. We also demonstrate that ALC attenuates mitochondrial fission in aged yeast cells, indicating a correlation between its protective effect and this process. Our findings suggest that ALC, used as therapeutic for stroke, myocardial infarction and neurodegenerative diseases, besides the well‐known anti‐oxidant effects, might exert protective effects also acting on mitochondrial morphology.     

High‐affinity cooperative Ca2+ binding by MICU1–MICU2 serves as an on–off switch for the uniporter

The mitochondrial calcium uniporter is a Ca²⁺‐activated Ca²⁺ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca²⁺ signals. It is regulated by two paralogous EF‐hand proteins—MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca²⁺. We reconstitute the MICU1–MICU2 heterodimer and demonstrate that it binds Ca²⁺ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria‐specific lipid cardiolipin. We determine the minimum Ca²⁺ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca²⁺‐binding affinity of MICU1–MICU2. We conclude that cooperative, high‐affinity interaction of the MICU1–MICU2 complex with Ca²⁺ serves as an on–off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca²⁺ signals.     

The ‘Expansion–Contraction’ model of Pleistocene biogeography: rocky shores suffer a sea change?

Approximately 20 000 years ago the last glacial maximum (LGM) radically altered the distributions of many Northern Hemisphere terrestrial organisms. Fewer studies describing the biogeographic responses of marine species to the LGM have been conducted, but existing genetic data from coastal marine species indicate that fewer taxa show clear signatures of post-LGM recolonization. We have assembled a mitochondrial DNA (mtDNA) data set for 14 co-distributed northeastern Pacific rocky-shore species from four phyla by combining new sequences from ten species with previously published sequences from eight species. Nuclear sequences from four species were retrieved from GenBank, plus we gathered new elongation factor 1-α sequences from the barnacle Balanus glandula . Results from demographic analyses of mtDNA for five (36%) species ( Evasterias troschelii, Pisaster ochraceus, Littorina sitkana, L. scutulata, Xiphister mucosus ) were consistent with large population expansions occurring near the LGM, a pattern expected if these species recently recolonized the region. However, seven (50%) species ( Mytilus trossulus, M. californianus, B. glandula, S. cariosus, Patiria miniata, Katharina tunicata , X. atropurpureus ) exhibited histories consistent with long-term stability in effective population size, a pattern indicative of regional persistence during the LGM. Two species of Nucella with significant mtDNA genetic structure showed spatially variable demographic histories. Multilocus analyses for five species were largely consistent with mtDNA: the majority of multilocus interpopulation divergence times significantly exceeded the LGM. Our results indicate that the LGM did not extirpate the majority of species in the northeastern Pacific; instead, regional persistence during the LGM appears a common biogeographic history for rocky-shore organisms in this region.     

Interaction of ‘toxic’ and ‘immunogenic’ A‐gliadin peptides with a membrane‐mimetic environment

Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in ‘toxic’ and ‘immunogenic’ classes, which elicit an innate immune response and an HLA‐mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31–43 (P31–43) and the 56–68 (P56–68) A‐gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane‐mimetic medium. We show that P31–43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56–68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD. Copyright © 2009 John Wiley & Sons, Ltd.     

From clear lakes to murky waters – tracing the functional response of high‐latitude lake communities to concurrent ‘greening’ and ‘browning’

Climate change and the intensification of land use practices are causing widespread eutrophication of subarctic lakes. The implications of this rapid change for lake ecosystem function remain poorly understood. To assess how freshwater communities respond to such profound changes in their habitat and resource availability, we conducted a space‐for‐time analysis of food‐web structure in 30 lakes situated across a temperature‐productivity gradient equivalent to the predicted future climate of subarctic Europe (temperature +3°C, precipitation +30% and nutrient +45 μg L^?1 total phosphorus). Along this gradient, we observed an increase in the assimilation of pelagic‐derived carbon from 25 to 75% throughout primary, secondary and tertiary consumers. This shift was overwhelmingly driven by the consumption of pelagic detritus by benthic primary consumers and was not accompanied by increased pelagic foraging by higher trophic level consumers. Our data also revealed a convergence of the carbon isotope ratios of pelagic and benthic food web endmembers in the warmest, most productive lakes indicating that the incorporation of terrestrial derived carbon into aquatic food webs increases as land use intensifies. These results, reflecting changes along a gradient characteristic of the predicted future environment throughout the subarctic, indicate that climate and land use driven eutrophication and browning are radically altering the function and fuelling of aquatic food webs in this biome.