RESUMEN
Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, "ER-to-Golgi" traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-ß-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.
Asunto(s)
Colesterol/metabolismo , Mezclas Complejas/metabolismo , ADN/metabolismo , Fosforilación/efectos de los fármacos , Esfingomielinas/metabolismo , Sistemas CRISPR-Cas , Citosol/metabolismo , Citosol/ultraestructura , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Aparato de Golgi/metabolismo , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Lipoilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Esfingomielina Fosfodiesterasa/metabolismo , beta-Ciclodextrinas/metabolismoRESUMEN
Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common architecture in all systems studied thus far, comprising three major components - the cytoplasmic sorting platform, envelope-spanning basal body and external needle with tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for these residues in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome.
Asunto(s)
Adenosina Trifosfatasas/ultraestructura , Proteínas Bacterianas/ultraestructura , Pseudomonas aeruginosa/ultraestructura , Sistemas de Secreción Tipo III/ultraestructura , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cuerpos Basales/enzimología , Cuerpos Basales/ultraestructura , Citoplasma/química , Citoplasma/genética , Citoplasma/ultraestructura , Citosol/ultraestructura , Transporte de Proteínas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Sistemas de Secreción Tipo III/química , Sistemas de Secreción Tipo III/genéticaRESUMEN
Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na+/K+-ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca2+-sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca2+ influx into the cytosol and that failing to pump Ca2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca2+ or Mn2+ uptake may be a way of controlling oleandrin toxicity.
Asunto(s)
Calcio/metabolismo , Cardenólidos/química , Glicósidos Cardíacos/química , Glicósidos Cardíacos/metabolismo , Manganeso/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Cardenólidos/farmacología , Glicósidos Cardíacos/farmacología , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Citosol/ultraestructura , Inhibidores Enzimáticos/metabolismo , Humanos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de FluorescenciaRESUMEN
Recent epidemiological studies link Periodontal disease(PD) to age-related macular degeneration (AMD). We documented earlier that Porphyromonas gingivalis(Pg), keystone oral-pathobiont, causative of PD, efficiently invades human gingival epithelial and blood-dendritic cells. Here, we investigated the ability of dysbiotic Pg-strains to invade human-retinal pigment epithelial cells(ARPE-19), their survival, intracellular localization, and the pathological effects, as dysfunction of RPEs leads to AMD. We show that live, but not heat-killed Pg-strains adhere to and invade ARPEs. This involves early adhesion to ARPE cell membrane, internalization and localization of Pg within single-membrane vacuoles or cytosol, with some nuclear localization apparent. No degradation of Pg or localization inside double-membrane autophagosomes was evident, with dividing Pg suggesting a metabolically active state during invasion. We found significant downregulation of autophagy-related genes particularly, autophagosome complex. Antibiotic protection-based recovery assay further confirmed distinct processes of adhesion, invasion and amplification of Pg within ARPE cells. This is the first study to demonstrate invasion of human-RPEs, begin to characterize intracellular localization and survival of Pg within these cells. Collectively, invasion of RPE by Pg and its prolonged survival by autophagy evasion within these cells suggest a strong rationale for studying the link between oral infection and AMD pathogenesis in individuals with periodontitis.
Asunto(s)
Autofagosomas , Autofagia , Infecciones por Bacteroidaceae , Citosol , Porphyromonas gingivalis , Epitelio Pigmentado de la Retina , Vacuolas , Autofagosomas/metabolismo , Autofagosomas/microbiología , Autofagosomas/ultraestructura , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Línea Celular , Citosol/metabolismo , Citosol/microbiología , Citosol/ultraestructura , Humanos , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/ultraestructura , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/microbiología , Epitelio Pigmentado de la Retina/ultraestructura , Vacuolas/microbiología , Vacuolas/patología , Vacuolas/ultraestructuraRESUMEN
Biomolecular condensation is a way of organizing cytosol in which proteins and nucleic acids coassemble into compartments. In the multinucleate filamentous fungus Ashbya gossypii, the RNA-binding protein Whi3 regulates the cell cycle and cell polarity through forming macromolecular structures that behave like condensates. Whi3 has distinct spatial localizations and mRNA targets, making it a powerful model for how, when, and where specific identities are established for condensates. We identified residues on Whi3 that are differentially phosphorylated under specific conditions and generated mutants that ablate this regulation. This yielded separation of function alleles that were functional for either cell polarity or nuclear cycling but not both. This study shows that phosphorylation of individual residues on molecules in biomolecular condensates can provide specificity that gives rise to distinct functional identities in the same cell.
Asunto(s)
Ciclo Celular/genética , Polaridad Celular/genética , Eremothecium/metabolismo , Proteínas Fúngicas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Alelos , Secuencia de Bases , Compartimento Celular/genética , Citosol/metabolismo , Citosol/ultraestructura , Eremothecium/genética , Eremothecium/ultraestructura , Proteínas Fúngicas/genética , Expresión Génica , Calor , Mutación , Fosforilación , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Estrés Fisiológico/genéticaRESUMEN
Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD+. PARP1 and PARP10 were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which PARP2 is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD+ precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.
Asunto(s)
Autofagia , Silenciador del Gen , Poli(ADP-Ribosa) Polimerasas/genética , Proteolisis , Adenilato Quinasa/metabolismo , Animales , Autofagia/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Cloroquina/farmacología , Medio de Cultivo Libre de Suero , Citosol/metabolismo , Citosol/ultraestructura , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Eliminación de Gen , Silenciador del Gen/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Desarrollo de Músculos/efectos de los fármacos , NAD/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteolisis/efectos de los fármacos , Sirtuina 1/metabolismoRESUMEN
The recently solved crystal structures of the human cysteine desulfurase NFS1, in complex with the LYR protein ISD11, the acyl carrier protein ACP, and the main scaffold ISCU, have shed light on the molecular interactions that govern initial cluster assembly on ISCU. Here, we aim to highlight recent insights into iron-sulfur (Fe-S) cluster (ISC) biogenesis in mammalian cells that have arisen from the crystal structures of the core ISC assembly complex. We will also discuss how ISCs are delivered to recipient proteins and the challenges that remain in dissecting the pathways that deliver clusters to numerous Fe-S recipient proteins in both the mitochondrial matrix and cytosolic compartments of mammalian cells.
Asunto(s)
Proteína Transportadora de Acilo/química , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión a Hierro/química , Hierro/química , Azufre/química , Proteína Transportadora de Acilo/metabolismo , Secuencia de Aminoácidos , Liasas de Carbono-Azufre/química , Biología Computacional , Cristalización , Citosol/metabolismo , Citosol/ultraestructura , Humanos , Proteínas de Unión a Hierro/metabolismo , Proteínas Reguladoras del Hierro/química , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Modelos Moleculares , Unión Proteica , Conformación Proteica , FrataxinaRESUMEN
BACKGROUND: Conjugation of triterpenoids such as betulinic acid 1 with the Triphenylphosphonium (TPP) group is a powerful approach to generating medicinal compounds. Their development proposes structure optimization in respect of availability and activity towards target cells and organelles. Selection of 1 or its precursor betulonic acid 2 and the optimal linker is of particular importance for drug candidate identification among the TPP-triterpenoid conjugates. OBJECTIVE: In this study, new C-28-TPP conjugated derivatives of 1 and 2 with the alkyl/alkoxyalkyl linkers of variable length were synthesized and compared regarding their anticancer, antibacterial, and mitochondriatargeted effects. METHODS: The TPP conjugates of 1 and 2 [6a-f, 7a-f] were synthesized by the reaction of halogenalkyl esters [3a-f, 4a-f, 5] with triphenylphosphine in acetonitrile upon heating. Cytotoxicity (MTT assay), antibacterial activity (microdilution assay), and mitochondrial effects (flow cytofluorometry) were studied. RESULTS: Conjugation with the TPP group greatly increased the cytotoxicity of the triterpenoids up to 30 times. The conjugates were up to 10-17 times more active against MCF-7 (IC50 = 0.17µM, 72h, 6c) and PC-3 (IC50 = 0.14µM, 72h, 6a) cancer cells than for human skin fibroblasts. The enhanced antibacterial (bactericidal) activity of the TPP-triterpenoid conjugates with MIC for Gram-positive bacteria as low as 2µM (6a, 7a) was for the first time revealed. The conjugates were found to effectively inhibit fluorescence of 2',7'-dichlorofluorescin probe in the cytosol upon oxidation, decrease transmembrane potential, and increase superoxide radical level in mitochondria. CONCLUSION: Relationships between the effects and structure of the TPP-triterpenoid conjugates were evaluated and discussed. Based on the results, 6a can be selected for further preclinical investigation as a potential anticancer compound.
Asunto(s)
Antibacterianos/síntesis química , Antineoplásicos/síntesis química , Ácido Oleanólico/análogos & derivados , Compuestos Organofosforados/síntesis química , Triterpenos Pentacíclicos/química , Alcanos/química , Antibacterianos/farmacología , Antineoplásicos/farmacología , Antioxidantes/síntesis química , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/ultraestructura , Diseño de Fármacos , Ésteres/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Bacterias Grampositivas/efectos de los fármacos , Halógenos/química , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Estructura Molecular , Ácido Oleanólico/química , Compuestos Organofosforados/farmacología , Células PC-3 , Relación Estructura-Actividad , Superóxidos/química , Superóxidos/metabolismo , Triterpenos/química , Ácido BetulínicoRESUMEN
The side effects induced by nanoparticle exposure at a cellular level are one of the priority research topics due to the steady increase in the use of nanoparticles (NPs). Recently, the focus on cellular morphology and mechanical behavior is gaining relevance in order to fully understand the cytotoxic mechanisms. In this regard, we have evaluated the morphomechanical alteration in human breast adenocarcinoma cell line (MCF-7) exposed to TiO2NPs at two different concentrations (25 and 50 µg/mL) and two time points (24 and 48 h). By using confocal and atomic force microscopy, we demonstrated that TiO2NP exposure induces significant alterations in cellular membrane elasticity, due to actin proteins rearrangement in cytoskeleton, as calculated in correspondence to nuclear and cytoplasmic compartments. In this work, we have emphasized the alteration in mechanical properties of the cellular membrane, induced by nanoparticle exposure.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citotoxinas/toxicidad , Nanopartículas/toxicidad , Titanio/toxicidad , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Fenómenos Biomecánicos , Membrana Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Citosol/ultraestructura , Citotoxinas/química , Elasticidad/efectos de los fármacos , Humanos , Células MCF-7 , Microscopía de Fuerza Atómica , Nanopartículas/ultraestructura , Titanio/químicaRESUMEN
We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins.
Asunto(s)
Tomografía con Microscopio Electrónico/métodos , Proteínas de la Membrana/ultraestructura , Quimiocina CCL7/metabolismo , Quimiocina CCL7/ultraestructura , Criopreservación/métodos , Citosol/metabolismo , Citosol/ultraestructura , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/ultraestructuraRESUMEN
Microorganisms adapt their biophysical properties in response to changes in their local environment. However, quantifying these changes at the single-cell level has only recently become possible, largely relying on fluorescent labeling strategies. In this work, we utilize yeast (Saccharomyces cerevisiae) to demonstrate label-free quantification of changes in both intracellular osmolarity and macromolecular concentration in response to changes in the local environment. By combining a digital holographic microscope with a millifluidic chip, the temporal response of cellular water flux was successfully isolated from the rate of production of higher molecular weight compounds, in addition to identifying the produced compounds in terms of the product of their refractive index increment [Formula: see text] and molar mass. The ability to identify, quantify and temporally resolve multiple biophysical processes in living cells at the single cell level offers a crucial complement to label-based strategies, suggesting broad applicability in studies of a wide-range of cellular processes.
Asunto(s)
Citosol/metabolismo , Saccharomyces cerevisiae/química , Análisis de la Célula Individual/métodos , Agua/metabolismo , Transporte Biológico , Citosol/química , Citosol/ultraestructura , Holografía , Dispositivos Laboratorio en un Chip , Concentración Osmolar , Presión Osmótica , Refractometría , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Análisis de la Célula Individual/instrumentación , Agua/químicaRESUMEN
Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility.
Asunto(s)
Carboxipeptidasas/genética , Infertilidad Masculina/genética , Microtúbulos/metabolismo , Procesamiento Proteico-Postraduccional , Espermátides/metabolismo , Espermatogénesis/genética , Tubulina (Proteína)/metabolismo , Animales , Carboxipeptidasas/deficiencia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Centriolos/metabolismo , Centriolos/patología , Centriolos/ultraestructura , Citosol/metabolismo , Citosol/ultraestructura , Ácido Glutámico/metabolismo , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Noqueados , Microtúbulos/patología , Microtúbulos/ultraestructura , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Espermátides/patología , Espermátides/ultraestructura , Tubulina (Proteína)/genéticaRESUMEN
The ESCRT machinery mediates membrane fission in a variety of processes in cells. According to current models, ESCRT-III proteins drive membrane fission by assembling into helical filaments on membranes. Here, we used 3D STORM imaging of endogenous ESCRT-III component IST1 to reveal the evolution of the structural organization of ESCRT-III in mammalian cytokinetic abscission. Using this approach, ESCRT-III ring and spiral assemblies were resolved and characterized at different stages of abscission. Visualization of IST1 structures in cells lacking the microtubule-severing enzyme spastin and in cells depleted of specific ESCRT-III components or the ATPase VPS4 demonstrated the contribution of these components to the organization and function of ESCRTs in cells. This work provides direct evidence that ESCRT-III proteins form helical filaments to mediate their function in cells and raises new mechanistic scenarios for ESCRT-driven cytokinetic abscission.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Citocinesis/genética , Citosol/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Microtúbulos/metabolismo , Proteínas Oncogénicas/genética , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citosol/ultraestructura , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Edición Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Microtúbulos/ultraestructura , Imagen Molecular , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Espastina/deficiencia , Espastina/genética , Imagen de Lapso de Tiempo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
The neural cell adhesion molecule (NCAM) is important for neural development and for plasticity in adult brain. Previous studies demonstrated a calmodulin-dependent import of a transmembrane fragment of NCAM into the nucleus that regulates gene expression. In a protein macroarray we identified importin-ß1 as a potential interaction partner of NCAM's cytoplasmic tail. The interaction was verified and an importin-ß1-dependent import of NCAM into the nucleus could be demonstrated using quantitative immunofluorescence analysis. Generation of NCAM deletion mutants revealed that the last amino acids of the cytoplasmic region of NCAM are dispensable whereas other parts of NCAM's cytoplasmic tail take part in its nuclear translocation. With this study we propose an alternative nuclear route for NCAM via the classical importin-mediated import.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Neuronas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/genética , Animales , Células COS , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular Tumoral , Núcleo Celular/ultraestructura , Chlorocebus aethiops , Citosol/ultraestructura , Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Neuronas/ultraestructura , Análisis por Matrices de Proteínas , Unión Proteica , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/genética , beta Carioferinas/genéticaRESUMEN
Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.
Asunto(s)
Técnicas Biosensibles , Polarización de Fluorescencia/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/metabolismo , Transducción de Señal , Análisis de la Célula Individual/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Color , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citosol/metabolismo , Citosol/ultraestructura , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Colorantes Fluorescentes/síntesis química , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Transfección , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo , Proteína Fluorescente RojaRESUMEN
Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions, we visualized autofluorescent protein fusions in both cell types. Nucleoprotein (N) and glycoprotein (G) localized to the nucleus and cytoplasm, phosphoprotein (P) was only found in the nucleus, and 3 (movement) and matrix (M) were present in the cytoplasm. This localization pattern is consistent with the model of nucleorhabdoviral replication of N, P, L and viral RNA forming a complex in the nucleus and the subvirion associating with M and then G during budding into perinuclear space. The comparable localization patterns in both organisms indicates a similar replication cycle. Changes in localization when proteins were co-expressed suggested viral proteins interact thus altering organelle targeting. We documented a limited number of direct protein interactions indicating host factors play a role in the virus protein interactions during the infection cycle.
Asunto(s)
Núcleo Celular/virología , Citosol/virología , Drosophila melanogaster/virología , Macrófagos/virología , Nicotiana/virología , Células Vegetales/virología , Rhabdoviridae/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Clonación Molecular/métodos , Citosol/metabolismo , Citosol/ultraestructura , Drosophila melanogaster/citología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Especificidad del Huésped , Interacciones Huésped-Patógeno , Macrófagos/metabolismo , Macrófagos/ultraestructura , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Imagen Óptica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Células Vegetales/metabolismo , Células Vegetales/ultraestructura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rhabdoviridae/crecimiento & desarrollo , Rhabdoviridae/metabolismo , Nicotiana/citología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the protein p62 in the formation of ALIS and demonstrated that two domains of p62-PB1 and UBA-are essential for ALIS assembly. Those two major binding domains of p62, also known as sequestosome 1, were shown to play a critical role in the formation of autophagosomes or cytoplasmic aggregates. Specifically, the PB1 domain is essential for self-oligomerization, and the UBA domain allows p62 to bind to polyubiquitin chains or ubiquitinated proteins. After stimulation of RAW 264.7 macrophages with lipopolysaccharide, we observed a significant decrease in the number of cells with ALIS. Importantly, cells overexpressing either a PB1 mutant or UBA-deleted p62 construct also exhibited a substantially diminished number of cells containing ALIS. Since both p62 and ubiquitin are found in ALIS, we evaluated the dynamics of YFP-tagged p62 in ALIS. In contrast to the findings of a previous study that evaluated GFP-tagged ubiquitin motility in ALIS, we determined that YFP-tagged p62 has very limited mobility. Lastly, we determined that GST-tagged full-length p62 binds to Lys-63-linked polyubiquitin chains but not to Lys-48-linked chains. Overall, our findings provide insight on the essential role that p62, particularly its PB1 and UBA domains, has in the formation of ALIS.
Asunto(s)
Citosol/ultraestructura , Poliubiquitina/metabolismo , Agregado de Proteínas/fisiología , Proteína Sequestosoma-1/química , Ubiquitina/metabolismo , Animales , Citosol/química , Humanos , Lipopolisacáridos/farmacología , Ratones , Unión Proteica , Dominios Proteicos , Células RAW 264.7RESUMEN
TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35 kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Neuronas , Oligonucleótidos/toxicidad , Isoformas de Proteínas/metabolismo , Línea Celular Transformada , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Citosol/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Inmunoprecipitación , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mutación/efectos de los fármacos , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Consumo de Oxígeno/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
Atherosclerosis develops as a consequence of inflammation and cell senescence. In critical factors involved in the atherosclerotic changes, reactive oxygen species (ROS) generation is considered a leading cause. While NADPH oxidases, particularly NOX2, are the main sources of ROS, how they are regulated in the disease is incompletely understood. In addition, how caveolae, the membrane structure implicated in oxLDL deposition under vascular endothelia, is involved in the oxLDL-mediated ROS production remains mostly elusive. We report here that macrophages exposed to oxLDL up-regulate its caveolin-1 expression, and the latter in turn up-regulates NOX2 p47phox level. This combination effect results in increased cellular senescence. Interestingly, oxLDL treatment causes the p47phox residing in the cytosol to translocate to the caveolae. Immunoprecipitation assays confirms that cavelin-1 is in high degree association with p47phox. These results suggest caveolin-1 may serve as the membrane target for p47phox and as a switch for ROS production following oxLDL exposure. Our results reveal a previously unknown molecular event in oxLDL-mediated cellular ageing, and may provide a target for clinical intervention for atherosclerosis.
Asunto(s)
Caveolas/efectos de los fármacos , Caveolina 1/genética , Lipoproteínas LDL/farmacología , NADPH Oxidasas/genética , Animales , Caveolas/metabolismo , Caveolas/ultraestructura , Caveolina 1/antagonistas & inhibidores , Caveolina 1/metabolismo , Senescencia Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Citosol/ultraestructura , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Transporte de Proteínas , Células RAW 264.7 , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Iron (Fe) is an essential metal involved in a wide spectrum of physiological functions. Sub-cellular characterization of the size, composition, and distribution of ferritin(iron) can provide valuable information on iron storage and transport in health and disease. In this study we employ magnetic force microscopy (MFM), transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS) to characterize differences in ferritin(iron) distribution and composition across injured and non-injured tissues by employing a rodent model of spinal cord injury (SCI). Our biophysical and ultrastructural analyses provide novel insights into iron distribution which are not obtained by routine biochemical stains. In particular, ferritin(iron) rich lysosomes revealed increased heterogeneity in MFM signal from tissues of SCI animals. Ultrastructural analysis using TEM elucidated that both cytosolic and lysosomal ferritin(iron) density was increased in the injured (spinal cord) and non-injured (spleen) tissues of SCI as compared to naïve animals. In-situ EELs analysis revealed that ferritin(iron) was primarily in Fe3+ oxidation state in both naïve and SCI animal tissues. The insights provided by this study and the approaches utilized here can be applied broadly to other systemic problems involving iron regulation or to understand the fate of exogenously delivered iron-oxide nanoparticles.