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BACKGROUND: Initiating discussion about death and dying is often considered a difficult topic for healthcare providers, thus there is a need for further research to understand this area, particularly in developing countries. The aim of this study was to describe preferences for the initiation of end-of-life care discussions in Indonesia, comparing the general population and health care professionals. METHODS: This cross-sectional, descriptive study analysed quantitative data from 368 respondents to an online questionnaire (255 general population (69%); 113 healthcare professionals (31%)) utilizing consecutive sampling and snowball sampling methods. RESULTS: Overall, most respondents (80%) stated that they would like to discuss end-of-life issues with a healthcare professional in the case of terminal illness. This was more marked amongst healthcare professionals compared with the general population (94% vs. 75%, respectively, p < 0,001). The preferred time for discussion was at first diagnosis (68% general population, 52% healthcare professionals, p = 0.017) and the preferred person to start the discussion was the doctor (59% general population, 71% healthcare professionals, p = 0.036). Fewer respondents wanted to know about prognosis compared to diagnosis (overall 76% v 93% respectively). CONCLUSION: Doctors have vital role in end-of-life care discussion, and attempts should be made to encourage physicians to initiate these conversations and respond to patient's requests when needed. These findings contribute to the existing body of knowledge in this area of practice, with focus on a developing country. The role of socio-cultural influences on these conversations warrants further research, in order to develop practical resources to support clinicians to appropriately conduct end-of-life care discussions with their patients and to provide data for policymakers to develop services.
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Cuidados Paliativos al Final de la Vida , Cuidado Terminal , Estudios Transversales , Humanos , Indonesia , Investigación CualitativaRESUMEN
Wallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinson's disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLDS and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders.
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Proteínas del Dominio Armadillo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Mitocondrias/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Degeneración Walleriana/metabolismo , Degeneración Walleriana/patología , Animales , Axones/metabolismo , Axones/patología , Drosophila , Masculino , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BLRESUMEN
Cancer therapeutics is a rapidly changing field which offers patients the prospect of a better quality of life and cure. Immunotherapy has become a unique approach for select metastatic solid tumours. While initial results do show durable responses in select patients, there are concerns on how best to utilise this expensive resource which can result in costly side effects and in whom the use of biomarkers to stratify patients is still in its infancy. Given the ageing population and extreme challenges on healthcare, economic modelling with regards to immunotherapy is imperative especially now when it is being considered for further cancer types.
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Inmunoterapia , Neoplasias/tratamiento farmacológico , Humanos , Inmunoterapia/efectos adversos , Inmunoterapia/economía , Inmunoterapia/métodos , Modelos Económicos , Neoplasias/psicología , Calidad de VidaRESUMEN
Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. Here, we assessed the axonal transport of different cargos in multiple Drosophila models of ALS. We found that axonal transport defects are common across all models tested, although they often showed a differential effect between mitochondria and vesicle cargos. Motor deficits were also common across the models and generally worsened with age, though surprisingly there was not a clear correlation between the severity of axonal transport defects and motor ability. These results further support defects in axonal transport as a common factor in models of ALS that may contribute to the pathogenic process.
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Esclerosis Amiotrófica Lateral/genética , Transporte Axonal/genética , Proteínas de Unión al ADN/genética , Proteínas/genética , Proteína FUS de Unión a ARN/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Axones/patología , Proteína C9orf72 , Proteínas de Unión al ADN/biosíntesis , Modelos Animales de Enfermedad , Drosophila/genética , Humanos , Larva/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mutación , Proteína FUS de Unión a ARN/biosíntesis , Superóxido Dismutasa-1/genéticaRESUMEN
BACKGROUND: Mitochondrial biogenesis is an essential process in all eukaryotes. Import of proteins from the cytosol into mitochondria is a key step in organelle biogenesis. Recent evidence suggests that a given mitochondrial protein does not take the same import route in all organisms, suggesting that pathways of mitochondrial protein import can be rewired through evolution. Examples of this process so far involve proteins destined to the mitochondrial intermembrane space (IMS). SCOPE OF REVIEW: Here we review the components, substrates and energy sources of the known mechanisms of protein import into the IMS. We discuss evolutionary rewiring of the IMS import routes, focusing on the example of the lactate utilisation enzyme cytochrome b2 (Cyb2) in the model yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. MAJOR CONCLUSIONS: There are multiple import pathways used for protein entry into the IMS and they form a network capable of importing a diverse range of substrates. These pathways have been rewired, possibly in response to environmental pressures, such as those found in the niches in the human body inhabited by C. albicans. GENERAL SIGNIFICANCE: We propose that evolutionary rewiring of mitochondrial import pathways can adjust the metabolic fitness of a given species to their environmental niche. This article is part of a Special Issue entitled Frontiers of Mitochondrial.
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Evolución Biológica , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Candida albicans/genética , Candida albicans/metabolismo , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The controlled biogenesis of mitochondria is a key cellular system coordinated with the cell division cycle, and major efforts in systems biology currently are directed toward understanding of the control points at which this coordination is achieved. Here we present insights into the function, evolution, and regulation of mitochondrial biogenesis through the study of the protein import machinery in the human fungal pathogen, Candida albicans. Features that distinguish C. albicans from baker's yeast (Saccharomyces cerevisiae) include the stringency of metabolic control at the level of oxygen consumption, the potential for ATP exchange through the porin in the outer membrane, and components and domains in the sorting and assembling machinery complex, a molecular machine that drives the assembly of proteins in the outer mitochondrial membrane. Analysis of targeting sequences and assays of mitochondrial protein import show that components of the electron transport chain are imported by distinct pathways in C. albicans and S. cerevisiae, representing an evolutionary rewiring of mitochondrial import pathways. We suggest that studies using this pathogen as a model system for mitochondrial biogenesis will greatly enhance our knowledge of how mitochondria are made and controlled through the course of the cell-division cycle.
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Evolución Biológica , Candida albicans/fisiología , Proteínas Portadoras/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Análisis por Conglomerados , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Cadenas de Markov , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Consumo de Oxígeno/fisiología , Filogenia , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae , Especificidad de la EspecieRESUMEN
Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly between the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Ca2+ and Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a signaling pathway underlying the subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise and activity-dependent regulation of mitochondria fission/fusion balance.
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Neuronas , Células Piramidales , Neuronas/metabolismo , Células Piramidales/fisiología , Hipocampo , Axones/metabolismo , Mitocondrias/metabolismo , Dendritas/fisiologíaRESUMEN
The apicomplexan parasite Cryptosporidium parvum possesses a mitosome, a relict mitochondrion with a greatly reduced metabolic capability. This mitosome houses a mitochondrial-type protein import apparatus, but elements of the protein import pathway have been reduced, and even lost, through evolution. The small Tim protein family is a case in point. The genomes of C. parvum and related species of Cryptosporidium each encode just one small Tim protein, CpTimS. This observation challenged the tenet that small Tim proteins are always found in pairs as α3ß3 hexamers. We show that the atypical CpTimS exists as a relatively unstable homohexamer, shedding light both on the early evolution of the small Tim protein family and on small Tim hexamer formation in contemporary eukaryotes.
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Proteínas Portadoras/química , Cryptosporidium/genética , Mitocondrias/genética , Chaperonas Moleculares/química , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cryptosporidium/química , Evolución Molecular , Mitocondrias/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Multimerización de Proteína , Subunidades de Proteína , Alineación de SecuenciaRESUMEN
Recent studies indicate that mitochondrial functions impinge on cell wall integrity, drug tolerance, and virulence of human fungal pathogens. However, the mechanistic aspects of these processes are poorly understood. We focused on the mitochondrial outer membrane SAM (Sorting and Assembly Machinery) complex subunit Sam37 in Candida albicans. Inactivation of SAM37 in C. albicans leads to a large reduction in fitness, a phenotype not conserved with the model yeast Saccharomyces cerevisiae. Our data indicate that slow growth of the sam37ΔΔ mutant results from mitochondrial DNA loss, a new function for Sam37 in C. albicans, and from reduced activity of the essential SAM complex subunit Sam35. The sam37ΔΔ mutant was hypersensitive to drugs that target the cell wall and displayed altered cell wall structure, supporting a role for Sam37 in cell wall integrity in C. albicans. The sensitivity of the mutant to membrane-targeting antifungals was not significantly altered. The sam37ΔΔ mutant was avirulent in the mouse model, and bioinformatics showed that the fungal Sam37 proteins are distant from their animal counterparts and could thus represent potential drug targets. Our study provides the first direct evidence for a link between mitochondrial function and cell wall integrity in C. albicans and is further relevant for understanding mitochondrial function in fitness, antifungal drug tolerance, and virulence of this major pathogen. Beyond the relevance to fungal pathogenesis, this work also provides new insight into the mitochondrial and cellular roles of the SAM complex in fungi.
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Candida albicans/metabolismo , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Candida albicans/patogenicidad , Candidemia/microbiología , Pared Celular/ultraestructura , Células Cultivadas , ADN Mitocondrial/metabolismo , Fluconazol/farmacología , Proteínas Fúngicas/genética , Hifa/metabolismo , Riñón/microbiología , Riñón/patología , Macrófagos/microbiología , Potencial de la Membrana Mitocondrial , Ratones , Pruebas de Sensibilidad Microbiana , Proteínas Mitocondriales/genética , Nematodos/microbiología , Forma de los Orgánulos , Fenotipo , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Homología de Secuencia de Aminoácido , VirulenciaRESUMEN
Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly in the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a new activity-dependent molecular mechanism underlying the extreme subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise regulation of mitochondria fission/fusion balance.
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Bacterial endosymbionts gave rise to mitochondria in a process that depended on the acquisition of protein import pathways. Modification and in some cases major re-tooling of the endosymbiont's cellular machinery produced these pathways, establishing mitochondria as organelles common to all eukaryotic cells. The legacy of this evolutionary tinkering can be seen in the homologies and structural similarities between mitochondrial protein import machinery and modern day bacterial proteins. Comparative analysis of these systems is revealing both possible routes for the evolution of the mitochondrial membrane translocases and a greater understanding of the mechanisms behind mitochondrial protein import. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
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Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Evolución Molecular , Humanos , Transporte de ProteínasRESUMEN
Mitochondria-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. Here, we identify and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid ß42 (Aß42). Together, our results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.
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Péptidos beta-Amiloides , Proteínas de Drosophila , Drosophila melanogaster , Retículo Endoplásmico , Mitocondrias , Fragmentos de Péptidos , Enfermedad de Alzheimer , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Animales , Senescencia Celular , Modelos Animales de Enfermedad , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Aptitud Genética , Locomoción/efectos de los fármacos , Longevidad/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Mitofagia/efectos de los fármacos , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/toxicidadRESUMEN
Dendritic calcium signaling is central to neural plasticity mechanisms that allow animals to adapt to the environment. Intracellular calcium release (ICR) from the endoplasmic reticulum has long been thought to shape these mechanisms. However, ICR has not been investigated in mammalian neurons in vivo. We combined electroporation of single CA1 pyramidal neurons, simultaneous imaging of dendritic and somatic activity during spatial navigation, optogenetic place field induction, and acute genetic augmentation of ICR cytosolic impact to reveal that ICR supports the establishment of dendritic feature selectivity and shapes integrative properties determining output-level receptive fields. This role for ICR was more prominent in apical than in basal dendrites. Thus, ICR cooperates with circuit-level architecture in vivo to promote the emergence of behaviorally relevant plasticity in a compartment-specific manner.
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Región CA1 Hipocampal/fisiología , Calcio/metabolismo , Dendritas/fisiología , Retículo Endoplásmico/metabolismo , Plasticidad Neuronal , Células de Lugar/fisiología , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Señalización del Calcio , Citosol/metabolismo , Electroporación , Femenino , Masculino , Ratones , Optogenética , Análisis de la Célula Individual , Navegación EspacialRESUMEN
A total of 16 different strains of Microbacterium spp. were isolated from contaminated soil and enriched on the carcinogen, hexavalent chromium [Cr(VI)]. The majority of the isolates (11 of the 16) were able to tolerate concentrations (0.1 mM) of cobalt, cadmium, and nickel, in addition to Cr(VI) (0.5-20 mM). Interestingly, these bacteria were also able to tolerate three different antibiotics (ranges: ampicillin 0-16 µg ml-1, chloramphenicol 0-24 µg ml-1, and vancomycin 0-24 µg ml-1). To gain genetic insight into these tolerance pathways, the genomes of these isolates were assembled and annotated. The genomes of these isolates not only have some shared genes (core genome) but also have a large amount of variability. The genomes also contained an annotated Cr(VI) reductase (chrR) that could be related to Cr(VI) reduction. Further, various heavy metal tolerance (e.g., Co/Zn/Cd efflux system) and antibiotic resistance genes were identified, which provide insight into the isolates' ability to tolerate metals and antibiotics. Overall, these isolates showed a wide range of tolerances to heavy metals and antibiotics and genetic diversity, which was likely required of this population to thrive in a contaminated environment.
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Mitochondrial Ca2+ uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca2+ uniporter has opened it up to genetic analysis in model organisms. Here, we report a comprehensive genetic characterization of all known uniporter components conserved in Drosophila. While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca2+ uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmentally lethal, consistent with unregulated Ca2+ uptake. Mutants for the neuronally restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. Our data reveal the interplay among components of the mitochondrial Ca2+ uniporter and shed light on their physiological requirements in vivo.
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Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Mutación , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , FenotipoRESUMEN
The pathogenic yeast Candida albicans escapes macrophages by triggering NLRP3 inflammasome-dependent host cell death (pyroptosis). Pyroptosis is inflammatory and must be tightly regulated by host and microbe, but the mechanism is incompletely defined. We characterized the C. albicans endoplasmic reticulum (ER)-mitochondrion tether ERMES and show that the ERMES mmm1 mutant is severely crippled in killing macrophages despite hyphal formation and normal phagocytosis and survival. To understand dynamic inflammasome responses to Candida with high spatiotemporal resolution, we established live-cell imaging for parallel detection of inflammasome activation and pyroptosis at the single-cell level. This showed that the inflammasome response to mmm1 mutant hyphae is delayed by 10 h, after which an exacerbated activation occurs. The NLRP3 inhibitor MCC950 inhibited inflammasome activation and pyroptosis by C. albicans, including exacerbated inflammasome activation by the mmm1 mutant. At the cell biology level, inactivation of ERMES led to a rapid collapse of mitochondrial tubular morphology, slow growth and hyphal elongation at host temperature, and reduced exposed 1,3-ß-glucan in hyphal populations. Our data suggest that inflammasome activation by C. albicans requires a signal threshold dependent on hyphal elongation and cell wall remodeling, which could fine-tune the response relative to the level of danger posed by C. albicans. The phenotypes of the ERMES mutant and the lack of conservation in animals suggest that ERMES is a promising antifungal drug target. Our data further indicate that NLRP3 inhibition by MCC950 could modulate C. albicans-induced inflammation. IMPORTANCE The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.
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Mitochondria fulfill central functions in cellular energetics, metabolism, and signaling. The outer membrane translocator complex (the TOM complex) imports most mitochondrial proteins, but its architecture is unknown. Using a cross-linking approach, we mapped the active translocator down to single amino acid residues, revealing different transport paths for preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel to recruit chaperones from the intermembrane space that guide the transfer of hydrophobic preproteins. The translocator contains three Tom40 ß-barrel channels sandwiched between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex exchanges with a dimeric isoform to assemble new TOM complexes. Dynamic coupling of α-helical receptors, ß-barrel channels, and chaperones generates a versatile machinery that transports about 1000 different proteins.