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Neurons receive correlated levels of excitation and inhibition, a feature that is important for proper brain function. However, how this relationship between excitatory and inhibitory inputs is established during the dynamic period of circuit wiring remains unexplored. Using multiple techniques, including in utero electroporation, electron microscopy, and electrophysiology, we reveal a tight correlation in the distribution of excitatory and inhibitory synapses along the dendrites of developing CA1 hippocampal neurons. This correlation was present within short dendritic stretches (<20 µm) and, surprisingly, was most pronounced during early development, sharply declining with maturity. The tight matching between excitation and inhibition was unexpected, as inhibitory synapses lacked an active zone when formed and exhibited compromised evoked release. We propose that inhibitory synapses form as a stabilizing scaffold to counterbalance growing excitation levels. This relationship diminishes over time, suggesting a critical role for a subcellular balance in early neuronal function and circuit formation.
Assuntos
Sinapses , Animais , Sinapses/metabolismo , Sinapses/fisiologia , Dendritos/metabolismo , Dendritos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Camundongos , Região CA1 Hipocampal/fisiologia , Região CA1 Hipocampal/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/metabolismo , Hipocampo/citologia , FemininoRESUMO
The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Complexos de Proteínas Captadores de Luz , Fotossíntese , Complexo de Proteína do Fotossistema II , Tilacoides , Tilacoides/metabolismo , Tilacoides/química , Complexos de Proteínas Captadores de Luz/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/química , Galactolipídeos/metabolismo , Galactolipídeos/química , LuzRESUMO
Here we introduce scattering-type scanning near-field optical microscopy (s-SNOM) as a novel tool for nanoscale chemical-imaging of sub-cellular organelles, nanomaterials and of the interactions between them. Our setup uses a tuneable mid-infrared laser and a sharp scanning probe to image at a resolution substantially surpassing the diffraction limit. The laser can be tuned to excite vibrational modes of functional groups in biomolecules, (e.g. amide moieties), in a way that enables direct chemical mapping without the need for labelling. We, for the first time, chemically image neuronal ultrastructure, identify neuronal organelles and sub-organelle structures as small as 10 nm and validate our findings using transmission electron microscopy (TEM). We produce chemical and morphological maps of neurons treated with gold nanospheres and characterize nanoparticle size and intracellular location, and their interaction with the plasma membrane. Our results show that the label-free nature of s-SNOM means it has a 'true' chemical resolution of up to 20 nm which can be further improved. We argue that it offers significant potential in nanomedicine for nanoscale chemical imaging of cell ultrastructure and the subcellular distribution of nanomaterials within tissues.
Assuntos
Nanopartículas , Nanoestruturas , Nanotecnologia/métodos , Microscopia/métodos , Nanoestruturas/química , LuzRESUMO
Traditional image acquisition for cryo focused ion-beam scanning electron microscopy (FIB-SEM) tomography often sees thousands of images being captured over a period of many hours, with immense data sets being produced. When imaging beam sensitive materials, these images are often compromised by additional constraints related to beam damage and the devitrification of the material during imaging, which renders data acquisition both costly and unreliable. Subsampling and inpainting are proposed as solutions for both of these aspects, allowing fast and low-dose imaging to take place in the Focused ion-beam scanning electron microscopy FIB-SEM without an appreciable loss in image quality. In this work, experimental data are presented which validate subsampling and inpainting as a useful tool for convenient and reliable data acquisition in a FIB-SEM, with new methods of handling three-dimensional data being employed in the context of dictionary learning and inpainting algorithms using a newly developed microscope control software and data recovery algorithm.
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Phase-change nanodroplets have attracted increasing interest in recent years as ultrasound theranostic nanoparticles. They are smaller compared to microbubbles and they may distribute better in tissues (e.g. in tumours). They are composed of a stabilising shell and a perfluorocarbon core. Nanodroplets can vaporise into echogenic microbubbles forming cavitation nuclei when exposed to ultrasound. Their perfluorocarbon core phase-change is responsible for the acoustic droplet vaporisation. However, methods to quantify the perfluorocarbon core in nanodroplets are lacking. This is an important feature that can help explain nanodroplet phase change characteristics. In this study, we fabricated nanodroplets using lipids shell and perfluorocarbons. To assess the amount of perfluorocarbon in the core we used two methods, 19F NMR and FTIR. To assess the cavitation after vaporisation we used an ultrasound transducer (1.1 MHz) and a high-speed camera. The 19F NMR based method showed that the fluorine signal correlated accurately with the perfluorocarbon concentration. Using this correlation, we were able to quantify the perfluorocarbon core of nanodroplets. This method was used to assess the content of the perfluorocarbon of the nanodroplets in solutions over time. It was found that perfluoropentane nanodroplets lost their content faster and at higher ratio compared to perfluorohexane nanodroplets. The high-speed imaging indicates that the nanodroplets generate cavitation comparable to that from commercial contrast agent microbubbles. Nanodroplet characterisation should include perfluorocarbon concentration assessment as critical information for their development.
Assuntos
Fluorocarbonos , Nanopartículas , Ultrassonografia , Nanopartículas/química , Volatilização , Meios de Contraste/química , Fluorocarbonos/química , MicrobolhasRESUMO
Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block-face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from postnatal day 22 [P22] to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared with neighboring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favor synchronous activity in CA1 networks.
Assuntos
Hipocampo , Terminações Pré-Sinápticas , Sinapses , Neurônios , DendritosRESUMO
Fluorescent InP-based quantum dots have emerged as valuable nanomaterials for display technologies, biological imaging, and optoelectronic applications. The inclusion of zinc can enhance both their emissive and structural properties and reduce interfacial defects with ZnS or CdS shells. However, the sub-particle distribution of zinc and the role this element plays often remains unclear, and it has previously proved challenging to synthesise Zn-alloyed InP-based nanoparticles using aminophosphine precursors. In this report, we describe the synthesis of alloyed InZnP using zinc carboxylates, achieving colour-tuneable fluorescence from the unshelled core materials, followed by a one-pot ZnS or CdS deposition using diethyldithiocarbamate precursors. Structural analysis revealed that the "core/shell" particles synthesised here were more accurately described as homogeneous extended alloys with the constituent shell elements diffusing through the entire core, including full-depth inclusion of zinc.
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Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type-specific and synapse type-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV+ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system.
Assuntos
Córtex Cerebral , Interneurônios , Biossíntese de Proteínas , Receptor ErbB-4 , Sinapses , Proteína 2 do Complexo Esclerose Tuberosa , Animais , Camundongos , Córtex Cerebral/metabolismo , Interneurônios/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sinapses/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
A challenge in neurology is the lack of efficient brain-penetrable neuroprotectants targeting multiple disease mechanisms. Plasmonic gold nanostars are promising candidates to deliver standard-of-care drugs inside the brain but have not been trialed as carriers for neuroprotectants. Here, we conjugated custom-made peptide dendrimers (termed H3/H6), encompassing motifs of the neurotrophic S100A4-protein, onto star-shaped and spherical gold nanostructures (H3/H6-AuNS/AuNP) and evaluated their potential as neuroprotectants and interaction with neurons. The H3/H6 nanostructures crossed a model blood-brain barrier, bound to plasma membranes, and induced neuritogenesis with the AuNS, showing higher potency/efficacy than the AuNP. The H3-AuNS/NP protected neurons against oxidative stress, the H3-AuNS being more potent, and against Parkinson's or Alzheimer's disease (PD/AD)-related cytotoxicity. Unconjugated S100A4 motifs also decreased amyloid beta-induced neurodegeneration, introducing S100A4 as a player in AD. Using custom-made dendrimers coupled to star-shaped nanoparticles is a promising route to activate multiple neuroprotective pathways and increase drug potency to treat neurodegenerative disorders.
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Doença de Alzheimer , Dendrímeros , Fármacos Neuroprotetores , Humanos , Fármacos Neuroprotetores/química , Peptídeos beta-Amiloides , Dendrímeros/farmacologia , Dendrímeros/uso terapêutico , Neurônios , Ouro/química , Doença de Alzheimer/tratamento farmacológicoRESUMO
Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of fundamental physiological processes. This signaling involves close physical contacts between the two organelles that are mediated by the VAPB-PTPIP51 â³tethering" proteins. The VAPB-PTPIP51 tethers facilitate inositol 1,4,5-trisphosphate (IP3) receptor delivery of Ca2+ from ER to mitochondria. Damage to the tethers is seen in Alzheimer's disease, Parkinson's disease and frontotemporal dementia with related amyotrophic lateral sclerosis (FTD/ALS). Understanding the mechanisms that regulate the VAPB-PTPIP51 interaction thus represents an important area of research. Recent studies suggest that an FFAT motif in PTPIP51 is key to its binding to VAPB but this work relies on in vitro studies with short peptides. Cellular studies to support this notion with full-length proteins are lacking. Here we address this issue. Immunoprecipitation assays from transfected cells revealed that deletion of the PTPIP51 FFAT motif has little effect on VAPB binding. However, mutation and deletion of a nearby coiled-coil domain markedly affect this binding. Using electron microscopy, we then show that deletion of the coiled-coil domain but not the FFAT motif abrogates the effect of PTPIP51 on ER-mitochondria contacts. Finally, we show that deletion of the coiled-coil domain but not the FFAT motif abrogates the effect of PTPIP51 on the IP3 receptor-mediated delivery of Ca2+ to mitochondria. Thus, the coiled-coil domain is essential for PTPIP51 ER-mitochondria signaling functions.
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Transient nuclear envelope ruptures during interphase (NERDI) occur due to cytoskeletal compressive forces at sites of weakened lamina, and delayed NERDI repair results in genomic instability. Nuclear envelope (NE) sealing is completed by endosomal sorting complex required for transport (ESCRT) machinery. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated protein BROX as a crucial factor required to accelerate repair of the NE. Critically, BROX binds Nesprin-2G, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2G ubiquitination and facilitates the relaxation of mechanical stress imposed by compressive actin fibers at the rupture site. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. Our results demonstrate that BROX coordinates mechanoregulation with membrane remodeling to ensure the maintenance of nuclear-cytoplasmic compartmentalization and genomic stability.
Assuntos
Núcleo Celular/fisiologia , Citoesqueleto/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/fisiologia , Actinas/química , Movimento Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos , Fenômenos Mecânicos , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.
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Antibacterianos/metabolismo , Iminas/metabolismo , Pseudomonas aeruginosa/genética , Piridinas/metabolismo , Transporte Biológico , Genes Bacterianos/genética , Testes de Sensibilidade Microbiana , Mutação , Pseudomonas aeruginosa/metabolismoRESUMO
Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.
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Tomografia com Microscopia Eletrônica , Elétrons , Microscopia Crioeletrônica , Congelamento , Humanos , Microscopia Eletrônica de VarreduraRESUMO
Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex "organization" of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.
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Efrina-B2/genética , Receptor EphB4/genética , Receptor EphB4/metabolismo , Válvulas Venosas/anormalidades , Válvulas Venosas/embriologia , Animais , Aorta/ultraestrutura , Comunicação Celular , Polaridade Celular , Proliferação de Células , Conexina 43/metabolismo , Conexinas/metabolismo , Endotélio , Efrina-B2/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutação , Fenótipo , Ultrassonografia , Malformações Vasculares/diagnóstico por imagem , Malformações Vasculares/genética , Insuficiência Venosa/diagnóstico por imagem , Válvulas Venosas/diagnóstico por imagem , Proteína alfa-4 de Junções ComunicantesRESUMO
Reliable antimicrobial susceptibility testing is essential in informing both clinical antibiotic therapy decisions and the development of new antibiotics. Mammalian cell culture media have been proposed as an alternative to bacteriological media, potentially representing some critical aspects of the infection environment more accurately. Here, we use a combination of NMR metabolomics and electron microscopy to investigate the response of Escherichia coli and Pseudomonas aeruginosa to growth in differing rich media to determine whether and how this determines metabolic strategies, the composition of the cell wall, and consequently susceptibility to membrane active antimicrobials including colistin and tobramycin. The NMR metabolomic approach is first validated by characterizing the expected E. coli acid stress response to fermentation and the accompanying changes in the cell wall composition, when cultured in glucose rich mammalian cell culture media. Glucose is not a major carbon source for P. aeruginosa but is associated with a response to osmotic stress and a modest increase in colistin tolerance. Growth of P. aeruginosa in a range of bacteriological media is supported by consumption of formate, an important electron donor in anaerobic respiration. In mammalian cell culture media, however, the overall metabolic strategy of P. aeruginosa is instead dependent on consumption of glutamine and lactate. Formate doping of mammalian cell culture media does not alter the overall metabolic strategy but is associated with polyamine catabolism, remodelling of both inner and outer membranes, and a modest sensitization of P. aeruginosa PAO1 to colistin. Further, in a panel of P. aeruginosa isolates an increase between 2- and 3-fold in sensitivity to tobramycin is achieved through doping with other organic acids, notably propionate which also similarly enhances the activity of colistin. Organic acids are therefore capable of nonspecifically influencing the potency of membrane active antimicrobials.
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Anti-Infecciosos , Pseudomonas aeruginosa , Parede Celular , Escherichia coli , Testes de Sensibilidade MicrobianaRESUMO
Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle. In human malarial infections, an antibody response specific for the Plasmodium falciparum protein PF3D7_1021800 was previously associated with protection against malaria, leading to an interest in PF3D7_1021800 as a candidate vaccine antigen. Antibodies to the protein were reported to inhibit egress, resulting in it being named schizont egress antigen-1 (SEA1). A separate study found that SEA1 undergoes phosphorylation in a manner dependent upon the parasite cGMP-dependent protein kinase PKG, which triggers egress. While these findings imply a role for SEA1 in merozoite egress, this protein has also been implicated in kinetochore function during schizont development. Therefore, the function of SEA1 remains unclear. Here, we show that P. falciparum SEA1 localizes in proximity to centromeres within dividing nuclei and that conditional disruption of SEA1 expression severely impacts the distribution of DNA and formation of merozoites during schizont development, with a proportion of SEA1-null merozoites completely lacking nuclei. SEA1-null schizonts rupture, albeit with low efficiency, suggesting that neither SEA1 function nor normal segmentation is a prerequisite for egress. We conclude that SEA1 does not play a direct mechanistic role in egress but instead acts upstream of egress as an essential regulator required to ensure the correct packaging of nuclei within merozoites.IMPORTANCE Malaria is a deadly infectious disease. Rationally designed novel therapeutics will be essential for its control and eradication. The Plasmodium falciparum protein PF3D7_1021800, annotated as SEA1, is under investigation as a prospective component of a malaria vaccine, based on previous indications that antibodies to SEA1 interfere with parasite egress from infected erythrocytes. However, a consensus on the function of SEA1 is lacking. Here, we demonstrate that SEA1 localizes to dividing parasite nuclei and is necessary for the correct segregation of replicated DNA into individual daughter merozoites. In the absence of SEA1, merozoites develop defectively, often completely lacking a nucleus, and, consequently, egress is impaired and/or aberrant. Our findings provide insights into the divergent mechanisms by which intraerythrocytic malaria parasites develop and divide. Our conclusions regarding the localization and function of SEA1 are not consistent with the hypothesis that antibodies against it confer protective immunity to malaria by blocking merozoite egress.
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Antígenos de Protozoários/genética , Eritrócitos/parasitologia , Merozoítos/genética , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/genética , Esquizontes/fisiologia , Antígenos de Protozoários/metabolismo , Divisão Celular , Humanos , Merozoítos/química , Fosforilação , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Estudos Prospectivos , Proteínas de Protozoários/metabolismoRESUMO
Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus. This process is known as mitochondrial retrograde response (MRR) and is induced by mitochondrial dysfunction. MRR results in the nuclear stabilization of prosurvival transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Here, we demonstrate that MRR is facilitated by contact sites between mitochondria and the nucleus. The translocator protein (TSPO) by preventing the mitophagy-mediated segregation o mitochonria is required for this interaction. The complex formed by TSPO with the protein kinase A (PKA), via the A-kinase anchoring protein acyl-CoA binding domain containing 3 (ACBD3), established the tethering. The latter allows for cholesterol redistribution of cholesterol in the nucleus to sustain the prosurvival response by blocking NF-κB deacetylation. This work proposes a previously unidentified paradigm in MRR: the formation of contact sites between mitochondria and nucleus to aid communication.