RESUMEN
Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Resistencia a la Enfermedad/inmunología , Inmunidad de la Planta , Transducción de Señal , Animales , Muerte Celular , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Ácido Glutámico/metabolismo , Membrana Dobles de Lípidos/metabolismo , Oocitos/metabolismo , Células Vegetales/metabolismo , Multimerización de Proteína , Protoplastos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Imagen Individual de Molécula , Vacuolas/metabolismo , XenopusRESUMEN
BACKGROUND: ß-adrenergic receptor (ß-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of ß-AR remains unclear. METHODS: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates ß-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the ß-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between ß-adrenergic insult and ß-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from ß-arrestin-1-S330A/S330D mutation and ß-adrenergic insult. RESULTS: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to ß-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted ß-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing ß-arrestin-1-S330D (active form) inhibited the ß-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. ß-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the ß-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS: AMPK phosphorylation of ß-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting ß-AR/cAMP/PKA activation. Subsequently, ß-arrestin-1 Ser330 phosphorylation blocks ß-AR-induced cardiac inflammasome activation and remodeling.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Isoproterenol , Miocitos Cardíacos , beta-Arrestina 1 , Animales , Fosforilación , beta-Arrestina 1/metabolismo , beta-Arrestina 1/genética , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Isoproterenol/toxicidad , Isoproterenol/farmacología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Ratones Endogámicos C57BL , Masculino , Receptores Adrenérgicos beta/metabolismo , Serina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Agonistas Adrenérgicos beta/farmacología , Agonistas Adrenérgicos beta/toxicidad , Células Cultivadas , Transducción de Señal , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , HumanosRESUMEN
Salmonella enterica, the etiological agent of gastrointestinal and systemic diseases, translocates a plethora of virulence factors through its type III secretion systems to host cells during infection. Among them, SpvB has been reported to harbor an ADP-ribosyltransferase domain in its C terminus, which destabilizes host cytoskeleton by modifying actin. However, whether this effector targets other host factors as well as the function of its N terminus still remains to be determined. Here, we found that SpvB targets clathrin and its adaptor AP-1 (adaptor protein 1) via interactions with its N-terminal domain. Notably, our data suggest that SpvB-clathrin/AP-1 associations disrupt clathrin-mediated endocytosis and protein secretion pathway as well. In addition, knocking down of AP-1 promotes Salmonella intracellular survival and proliferation in host cells.
Asunto(s)
Salmonella enterica , Salmonella typhimurium , Salmonella typhimurium/metabolismo , Factor de Transcripción AP-1/metabolismo , Salmonella enterica/metabolismo , Factores de Virulencia/metabolismo , Actinas/metabolismo , Clatrina/metabolismoRESUMEN
Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.
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Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Auxilinas/metabolismo , Células COS , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Invaginaciones Cubiertas de la Membrana Celular/química , Endosomas/química , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/análisis , Fosfatidilinositoles/química , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Fosfotransferasas/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Animales , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/genética , Reparación del ADN por Recombinación , ADN , Mamíferos/genéticaRESUMEN
The dynamics and stoichiometry of receptors newly delivered on the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-ß type II receptor (TßRII), a typical serine/threoninekinase receptor, was studied with this method. We first eliminated the fluorescence signals from the pre-existing EGFP-labeled TßRII molecules on the plasma membrane, and then we recorded the individual newly appeared TßRII-GFP by total-internal-reflection fluorescence imaging. The fluorescence-intensity distributions, photobleaching steps, and diffusion rates of the single TßRII-GFP molecules were analyzed. We reported, for the first time, that TßRII was transported to the plasma membrane mainly in the monomeric form in both resting and TGF-ß1stimulated cells. This strongly supported our former discovery that TßRII could exist as a monomer on the cell membrane. We also found that ligand stimulation resulted in enhanced delivery rates and prolonged membrane-association times for the TßRII molecules. On the basis of these observations, we proposed a mechanism of TGF-ß1-induced TßRII dimerization for receptor activation. Our method provides a useful tool for the real-time quantification of the spatial arrangement, mobility, and oligomerization of cell-surface proteins in living cells, thus providing a better understanding of cell signaling.
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Membrana Celular/metabolismo , Imagen Óptica , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Imagen Individual de Molécula , Membrana Celular/química , Células HeLa , Humanos , Receptores de Factores de Crecimiento Transformadores beta/análisis , Células Tumorales CultivadasRESUMEN
UNLABELLED: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.
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Antivirales/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Tiourea/análogos & derivados , Internalización del Virus/efectos de los fármacos , Actinas/metabolismo , Animales , Línea Celular , Células Cultivadas , Clatrina/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/virología , Técnicas de Inactivación de Genes , Fiebre Hemorrágica Americana/genética , Fiebre Hemorrágica Americana/metabolismo , Fiebre Hemorrágica Americana/virología , Humanos , Virus Junin/efectos de los fármacos , Virus Junin/fisiología , Ratones , Unión Proteica , Transporte de Proteínas , Proteolisis , Ribonucleoproteínas/metabolismo , Tiourea/farmacología , Carga Viral , Proteínas Virales/metabolismo , Acoplamiento Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Actin and dynamin work cooperatively to drive the invagination and scission of clathrin-coated pits (CCPs). However, little is known about the mechanism that orchestrates the spatiotemporal recruitment of dynamin and actin. Here, we have identified the mammalian actin-binding protein 1 (mAbp1; also called HIP-55 or SH3P7), which could bind to clathrin, actin, as well as dynamin, as an adaptor that links the dynamic recruitment of dynamin and actin for the scission of CCPs. Live-cell imaging reveals that mAbp1 is specifically recruited at a late stage of the long-lived CCPs. mAbp1 knockdown impaired CCP scission by reducing dynamin recruitment at the plasma membrane. However, actin disruption remarkably eliminates mAbp1 recruitment and thus dynamin recruitment. These data suggest that by binding to both clathrin and F-actin, mAbp1 is specifically recruited at a late stage of CCP formation, which subsequently recruits dynamin to CCPs.
Asunto(s)
Actinas/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Dinaminas/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Línea Celular Tumoral , Clatrina/genética , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Dinaminas/genética , Humanos , Immunoblotting , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Microscopía Electrónica de Transmisión , Microscopía Fluorescente/métodos , Células 3T3 NIH , Unión Proteica , Imagen de Lapso de Tiempo/métodos , Dominios Homologos src/genéticaRESUMEN
OBJECTIVE: The aims of the study were to retrospectively evaluate the imaging characteristics of cavernous sinus angioleiomyoma and explore appropriate surgical approaches. METHODS: A total of four cases admitted to our hospital for repeated episodes of headache were enrolled. The common clinical manifestations included decreased vision, diplopia, blepharoptosis and facial numbness. The first three patients were misdiagnosed with cavernous sinus hemangioma, meningioma and chordoma, respectively. The fourth case was suspected to be angioleiomyoma. Imaging examination showed an oval-shaped tumor occupying the cavernous sinus. The tumor was hypointense on T1-weighted magnetic resonance imaging and extremely hyperintense on T2-weighted scan, which was similar to the cerebrospinal fluid, and was progressively enhanced from one side after gadolinium injection, pushing the internal carotid artery toward the inner side. RESULTS: Surgical resection was performed via an epidural approach for these four cases. Clinical symptoms were improved after surgery and no recurrence was observed during follow-up visits (average, 47.5 months). CONCLUSIONS: The initial symptom of cavernous sinus angioleiomyoma was repeated headache. The tumor seems extremely hyperintense on a T2-weighted image and expansive growth is pushing away the internal carotid artery rather than encasing it. It was progressively enhanced from one side after gadolinium injection. Surgical treatment based on an epidural approach had an excellent outcome in tumor resection and nerve protection.
Asunto(s)
Angiomioma/diagnóstico , Angiomioma/cirugía , Seno Cavernoso , Neoplasias de Tejido Vascular/diagnóstico , Neoplasias de Tejido Vascular/cirugía , Adulto , Angiomioma/complicaciones , Blefaroptosis/etiología , Seno Cavernoso/patología , Seno Cavernoso/cirugía , Femenino , Cefalea/etiología , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Neoplasias de Tejido Vascular/complicaciones , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Trastornos de la Visión/etiologíaRESUMEN
BACKGROUND: By definition, a cavernous sinus is a vascular plexus. A hemangioma is a vascular tumor which is rich in vascular plexus and caused by vascular deformity or tumor-like proliferation. Radiosurgery can effectively control the growth of smaller cavernous sinus hemangiomas (CSHs). This paper aimed to quantify the MRI characteristics of CSHs which provided a basis for further treatment. METHODS: A retrospective analysis was conducted of the MRI characteristics of 133 cavernous sinus lesion patients between January 2006 and December 2009, and none of them was diagnosed without histology. Two groups were set apart according to the pathological results: a CSH group (24 patients) and a non-CSH group (109 patients). The latter was further divided into 3 categories: a meningioma group (33 patients), a neurilemmoma group (37 patients) and a miscellaneous group (39 patients). A single-factor χ(2) analysis was performed to analyze the data on the 12 MRI characteristics. RESULTS: T2-weighted ultrahigh signal, signal uniformity, dumbbell-like appearance and infiltration of the sellar region were considered statistically significant for the differentiation of CSHs from non-CSHs. If these 4 items served as the diagnostic criteria, the sensitivity, specificity and accuracy was 87.5, 96.3 and 94.7%, respectively. CONCLUSION: Smaller CSHs can be treated by radiosurgery directly, without performance of a pathological biopsy.
Asunto(s)
Seno Cavernoso/patología , Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/patología , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Imagen por Resonancia Magnética/métodos , Neoplasias del Sistema Nervioso Central/cirugía , Hemangioma Cavernoso del Sistema Nervioso Central/cirugía , Humanos , Meningioma/diagnóstico , Meningioma/patología , Meningioma/cirugía , Neurilemoma/diagnóstico , Neurilemoma/patología , Neurilemoma/cirugía , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: In bypass surgery for moyamoya disease (MMD), the superficial temporal artery's (STA) pressure needs to surpass that of the cortical M4 recipient of the middle cerebral artery (MCA), boosting cerebral blood flow into the MCA and enhancing cerebral circulation. This study investigates the STA-MCA arterial pressure parameters and gradients during bypass surgery, aiming to deepen our understanding of hemodynamic shifts pre- and post-operation. METHODS: DSA imaging data were prospectively collected from patients diagnosed with bilateral MMD who underwent STA-MCA bypass surgery between 2022 and 2023 and stratified according to the Suzuki stage. The mean arterial pressure (MAP) of the donor and recipient arteries was directly measured during the STA-MCA bypass procedure, and these data were statistically analyzed and evaluated. RESULTS: Among 48 MMD patients, Suzuki grading revealed that 43.8% were in early stages (II and III), while 56.2% were in advanced stages (IV, V, and VI). Predominantly, 77.1% presented with ischemic-type MMD and 22.9% with hemorrhagic type. Pre-bypass assessments showed that 62.5% exhibited antegrade blood flow direction, and 37.5% had retrograde. The mean recipient artery pressure was 35.0 ± 2.3 mmHg, with a mean donor-recipient pressure gradient (δP) of 46.4 ± 2.5 mmHg between donor and recipient arteries. Post-bypass, mean recipient artery pressure increased to 73.3 ± 1.6 mmHg. No significant correlation (r = 0.18, P = 0.21) was noted between δP and Suzuki staging. CONCLUSION: Our study elucidated that cerebral blood pressure significantly decreases beyond the moyamoya network at the distal M4 segment. Furthermore, we observed bidirectional flow in MCA territories and a significant positive pressure gradient between the STA and M4 segments. The lack of correlation between Suzuki stages and M4 pressures indicates that angiographic severity may not reflect hemodynamic conditions before surgery, highlighting the need for customized surgical approaches.
RESUMEN
Mutations of TRAPPC12 are associated with progressive childhood encephalopathy including abnormal white matter. However, the underlying pathogenesis is still unclear. Here, we found that Trappc12 deficiency in CG4 and oligodendrocyte progenitor cells (OPCs) affects their differentiation and maturation. In addition, TRAPPC12 interacts with Mea6/cTAGE5, and Mea6/cTAGE5 ablation in OPCs affects their proliferation and differentiation, leading to marked hypomyelination, compromised synaptic functionality, and aberrant behaviors in mice. We reveal that TRAPPC12 is associated with COPII components at ER exit site, and Mea6/cTAGE5 cKO disrupts the trafficking pathway by affecting the distribution and/or expression of TRAPPC12, SEC13, SEC31A, and SAR1. Moreover, we observed marked disturbances in the secretion of pleiotrophin (PTN) in Mea6-deficient OPCs. Notably, exogenous PTN supplementation ameliorated the differentiation deficits of these OPCs. Collectively, our findings indicate that the association between TRAPPC12 and MEA6 is important for cargo trafficking and white matter development.
RESUMEN
Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α-SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α-XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α-SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.
Asunto(s)
Estrés del Retículo Endoplásmico , Endorribonucleasas , Proteínas Serina-Treonina Quinasas , Proteína 1 de Unión a la X-Box , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Humanos , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Animales , Empalme del ARN , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/genética , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Factores de Transcripción del Factor Regulador X/metabolismo , Factores de Transcripción del Factor Regulador X/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Respuesta de Proteína Desplegada , Ratones , Células HeLa , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/genética , Transducción de SeñalRESUMEN
Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.
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Microscopía Fluorescente/métodos , Algoritmos , Animales , Membrana Celular/metabolismo , Difusión , Colorantes Fluorescentes/farmacología , Humanos , Cinética , Microscopía Confocal/métodos , Modelos Biológicos , Nanotecnología/métodos , Receptores de Formil Péptido/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Prevention of rebleeding plays an important role in the treatment of hemorrhagic moyamoya disease, because rebleeding results in high mortality and morbidity. We discuss possible treatment for patients with moyamoya disease accompanied with distal choroidal artery aneurysms and review the literature to summarize clinical treatment and mechanisms. The cases of three male patients who suffered from intraventricular hemorrhage are presented. Computed tomography (CT) and digital subtractive angiography (DSA) revealed that bleeding was believed to be caused by ruptured aneurysms originating from distal choroidal artery aneurysms. Two patients successfully underwent superficial temporal artery (STA)-middle cerebral artery (MCA) bypass combined with encephalo-duro-myo-synangiosis (EDMS) and the obliteration of the aneurysm. The follow-up DSA or CT scan demonstrated that the aneurysms completely disappeared with the patency of the reconstructed artery. Neither of the patients experienced rebleeding during the follow-up period (up to 34 months). Given conservative treatment, the third patient experienced recurrent hemorrhages 4 months after the first ictus. This study describes treatment for moyamoya disease accompanied with distal choroidal artery aneurysms. Our experience suggests that cerebral revascularization combined with obliteration of the complicated distal aneurysm in the same session is a possible treatment.
Asunto(s)
Aneurisma Roto/cirugía , Arterias Cerebrales/cirugía , Aneurisma Intracraneal/cirugía , Enfermedad de Moyamoya/cirugía , Adulto , Aneurisma Roto/complicaciones , Aneurisma Roto/patología , Angiografía de Substracción Digital , Arterias Cerebrales/patología , Humanos , Aneurisma Intracraneal/complicaciones , Aneurisma Intracraneal/patología , Masculino , Persona de Mediana Edad , Enfermedad de Moyamoya/complicaciones , Enfermedad de Moyamoya/patología , Complicaciones Posoperatorias , Pronóstico , Literatura de Revisión como Asunto , Tomografía Computarizada por Rayos XRESUMEN
OBJECTIVE: Cavernous sinus hemangiomas (CSHs) are infrequent benign neoplasms. This meta-analysis was conducted with the objective of examining the prognostic outcomes of surgical interventions and radiotherapy (RT) in patients diagnosed with CSHs. METHODS: A comprehensive literature search was performed across PubMed, Embase, and Web of Science databases, with traceability up to June 22, 2021. The evaluation of continuous variables was conducted by applying the weighted mean difference (WMD) and 95% confidence interval. A one-arm meta-analysis was used to scrutinize the tumor control rate, clinical improvement rate, recovery rates of abducens nerve palsy and visual disturbance, total resection rate, and the incidence rate of permanent nerve palsy post-treatment. RESULTS: In total, 29 articles were incorporated into the meta-analysis. Post-RT for CSHs, a significant reduction in tumor volume was observed (WMD [95% confidence interval] = -17.16 [-21.52, -12.80] cm3). The tumor control rate, clinical improvement rate, recovery rate of abducens nerve palsy, and the recovery rate of visual disturbance were 97.1% (92.9, 99.7), 91.9% (82.3, 98.5), 95.6% (83.2, 100.0), and 86.3% (65.0, 99.5), respectively. Following surgical treatment, the total resection rate, mean intraoperative blood loss, recovery rate of visual disturbance, incidence rate of permanent nerve palsy, and recovery rate of abducens nerve palsy were 73.2% (57.1, 86.9), 971.17 mL (584.07, 1358.27), 66.4% (32.4, 0.942), 16.0% (4.6, 31.1), and 70.6% (51.0, 87.7), respectively. Notably, the recovery rate of abducens nerve palsy post-RT was markedly higher than postsurgical treatment. CONCLUSIONS: The results of this meta-analysis underscore that RT is an effective and safe treatment modality for CSHs. Furthermore, the prognostic outcomes of RT demonstrated superiority over surgical intervention.
RESUMEN
HER2 belongs to the human epidermal growth factor receptor tyrosine kinase family. Its overexpression or hyperactivation is a leading cause for multiple types of cancers. HER2 functions mainly through dimerization with other family members, such as EGFR. However, the molecular details for heterodimer assembly have not been completely understood. Here, we report cryo-EM structures of the EGF- and epiregulin-bound EGFR/HER2 ectodomain complexes at resolutions of 3.3 Å and 4.5 Å, respectively. Together with the functional analyses, we demonstrate that only the dimerization arm of HER2, but not that of EGFR, is essential for their heterodimer formation and signal transduction. Moreover, we analyze the differential membrane dynamics and transient interactions of endogenous EGFR and HER2 molecules in genome-edited cells using single-molecule live-cell imaging. Furthermore, we show that the interaction with HER2 could allow EGFR to resist endocytosis. Together, this work deepens our understanding of the unique structural properties and dynamics of the EGFR/HER2 complex.
RESUMEN
The goal when imaging bioprocesses with optical microscopy is to acquire the most spatiotemporal information with the least invasiveness. Deep neural networks have substantially improved optical microscopy, including image super-resolution and restoration, but still have substantial potential for artifacts. In this study, we developed rationalized deep learning (rDL) for structured illumination microscopy and lattice light sheet microscopy (LLSM) by incorporating prior knowledge of illumination patterns and, thereby, rationally guiding the network to denoise raw images. Here we demonstrate that rDL structured illumination microscopy eliminates spectral bias-induced resolution degradation and reduces model uncertainty by five-fold, improving the super-resolution information by more than ten-fold over other computational approaches. Moreover, rDL applied to LLSM enables self-supervised training by using the spatial or temporal continuity of noisy data itself, yielding results similar to those of supervised methods. We demonstrate the utility of rDL by imaging the rapid kinetics of motile cilia, nucleolar protein condensation during light-sensitive mitosis and long-term interactions between membranous and membrane-less organelles.
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Aprendizaje Profundo , Microscopía Fluorescente/métodos , Redes Neurales de la ComputaciónRESUMEN
Cells living in geometrically confined microenvironments are ubiquitous in various physiological processes, e.g., wound closure. However, it remains unclear whether and how spatially geometric constraints on host cells regulate bacteria-host interactions. Here, we reveal that interactions between bacteria and spatially constrained cell monolayers exhibit strong spatial heterogeneity, and that bacteria tend to adhere to these cells near the outer edges of confined monolayers. The bacterial adhesion force near the edges of the micropatterned monolayers is up to 75 nN, which is ~3 times higher than that at the centers, depending on the underlying substrate rigidities. Single-cell RNA sequencing experiments indicate that spatially heterogeneous expression of collagen IV with significant edge effects is responsible for the location-dependent bacterial adhesion. Finally, we show that collagen IV inhibitors can potentially be utilized as adjuvants to reduce bacterial adhesion and thus markedly enhance the efficacy of antibiotics, as demonstrated in animal experiments.
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Adhesión Bacteriana , Colágeno , Animales , Adhesión Bacteriana/fisiología , Colágeno/metabolismo , Fenómenos Mecánicos , Bacterias/metabolismo , Adhesión CelularRESUMEN
This study provides a parallel cytotoxicity study of water-soluble single-walled carbon nanotubes and fullerene C70(C(COOH)2)4-8 on Nicotiana tabacum BY-2 cells. After BY-2 cells were treated with single-walled carbon nanotubes or C70(C(COOH)2)4-8, the cells adopted a shrunken morphology and exhibited lower proliferation rate. Fluorescence imaging of the treated cells revealed increased reactive oxygen species generation, abnormal mitochondrial distribution, decreased mitochondrial activity, and impaired actin cytoskeleton arrangement. However, no obvious cells death was induced by either single-walled carbon nanotubes or C70(C(COOH)2)4-8. Compared with single-walled carbon nanotubes, C70(C(COOH)2)4-8 exerted more serious adverse effects on BY-2 cells.