RESUMEN
Coherent Raman microscopy has become a powerful tool in label-free, non-destructive and fast cell imaging. Here we apply high spectral resolution multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy in the high wavenumber region to the study of the cell cycle. We show that heterochromatin - the condensed state of chromatin - can be visualised by means of the vibrational signature of proteins taking part in its condensation. Thus, we are able to identify chromosomes and their movement during mitosis, as well as structures like nucleoli and nuclear border in interphase. Furthermore, the specific organization of the endoplasmic reticulum during mitosis is highlighted. Finally, we stress that MCARS can reveal the biochemical impact of the fixative method at the cellular level. Beyond the study of the cell cycle, this work introduces a label-free imaging approach that enables the visualization of cellular processes where chromatin undergoes rearrangements.
Asunto(s)
Cromatina/ultraestructura , Ciclo Celular , Nucléolo Celular/ultraestructura , Cromatina/química , Retículo Endoplásmico/ultraestructura , Citometría de Flujo , Células HEK293/ultraestructura , Humanos , Interfase , Mitosis , Espectrometría Raman/métodosRESUMEN
No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.
Asunto(s)
Antipsicóticos/uso terapéutico , Aripiprazol/uso terapéutico , Ataxina-3/metabolismo , Enfermedad de Machado-Joseph/tratamiento farmacológico , Enfermedad de Machado-Joseph/metabolismo , Proteínas Mutantes/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Ataxina-3/genética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/ultraestructura , Modelos Animales de Enfermedad , Drosophila , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Células HEK293/ultraestructura , Humanos , Enfermedad de Machado-Joseph/genética , Ratones , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Cultivo de Órganos , Péptidos/genética , Piperidinas/farmacología , Piranos/farmacología , Pirazoles/farmacologíaRESUMEN
The function of the hepatitis B virus X protein (HBx) has been investigated in hepatoma cell lines before; however, its function in the canonical HEK 293 cell line has not been addressed. In this study, we found that HBx increased cellular interaction by fusing the gap between HEK 293 cells, which is different from what has been reported previously. We also found that HBx enhanced the expression of E-cadherin in hepatoma cell lines instead of decreasing it as reported previously. The increase in E-cadherin was mediated by the enhanced levels of Src, which also differs from previous reports. Finally, we observed that HBx can accelerate cell growth by increasing the percentage of cells that are positioned at the division stage. Further analysis showed that the increased growth was caused by increased CDK4 expression and Ki67(+) populations. Additionally, reduced apoptosis was found in HEK 293 cells expressing HBx due to an increase in the anti-apoptotic protein-Bcl2. Collectively, the different functions of HBx in HEK 293 cells suggest that its role is cell dependent.
Asunto(s)
Células HEK293/virología , Transactivadores/fisiología , Apoptosis/fisiología , Cadherinas/metabolismo , Células HEK293/ultraestructura , Virus de la Hepatitis B/fisiología , Humanos , Proteínas Reguladoras y Accesorias ViralesRESUMEN
PURPOSE: Mutations in BEST1, encoding bestrophin-1 (Best1), cause autosomal recessive bestrophinopathy (ARB). Encoding bestrophin-1 is a pentameric anion channel localized to the basolateral plasma membrane of the RPE. Here, we characterize the effects of the mutations R141H (CGC > CAC) and I366fsX18 (c.1098_1100+7del), identified in a patient in our practice, on Best1 trafficking, oligomerization, and channel activity. METHODS: Currents of Cl- were assessed in transfected HEK293 cells using whole-cell patch clamp. Best1 localization was assessed by confocal microscopy in differentiated, human-induced pluripotent stem cell-derived RPE (iPSC-RPE) cells following expression of mutants via adenovirus-mediated gene transfer. Oligomerization was evaluated by coimmunoprecipitation in iPSC-RPE and MDCK cells. RESULTS: Compared to Best1, Best1 I366fsX18 currents were increased while Best1 R141H Cl- currents were diminished. Coexpression of Best1 R141H with Best1 or Best1 I366fsX18 resulted in rescued channel activity. Overexpressed Best1, Best1 R141H, and Best1 I366fsX18 were all properly localized in iPSC-RPE cells; Best1 R141H and Best1 I366fsX18 coimmunoprecipitated with endogenous Best1 in iPSC-RPE cells and with each other in MDCK cells. CONCLUSIONS: The first 366 amino acids of Best1 are sufficient to mediate channel activity and homo-oligomerization. The combination of Best1 and Best1 R141H does not cause disease, while Best1 R141H together with Best1 I366fsX18 causes ARB. Since both combinations generate comparable Cl- currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1 I366fsX18 differs from Best1 in that it lacks most of the cytosolic C-terminal domain, suggesting that the loss of this region contributes significantly to the pathogenesis of ARB in this patient.
Asunto(s)
Canales de Cloruro/genética , ADN/genética , Enfermedades Hereditarias del Ojo/genética , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Mutación , Enfermedades de la Retina/genética , Epitelio Pigmentado de la Retina/ultraestructura , Adolescente , Bestrofinas , Western Blotting , Membrana Celular/metabolismo , Canales de Cloruro/biosíntesis , Canales de Cloruro/metabolismo , Análisis Mutacional de ADN , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Hereditarias del Ojo/patología , Proteínas del Ojo/biosíntesis , Femenino , Angiografía con Fluoresceína , Fondo de Ojo , Genes Recesivos , Células HEK293/metabolismo , Células HEK293/ultraestructura , Humanos , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Técnicas de Placa-Clamp , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
The nucleus is a critical subcellular compartment for the pathogenesis of polyglutamine disorders, including Huntington's disease (HD). Recent studies suggest the first 17-amino-acid domain (N17) of mutant huntingtin (mHTT) mediates its nuclear exclusion in cultured cells. Here, we test whether N17 could be a molecular determinant of nuclear mHTT pathogenesis in vivo. BAC transgenic mice expressing mHTT lacking the N17 domain (BACHD-ΔN17) show dramatically accelerated mHTT pathology exclusively in the nucleus, which is associated with HD-like transcriptionopathy. Interestingly, BACHD-ΔN17 mice manifest more overt disease-like phenotypes than the original BACHD mice, including body weight loss, movement deficits, robust striatal neuron loss, and neuroinflammation. Mechanistically, N17 is necessary for nuclear exclusion of small mHTT fragments that are part of nuclear pathology in HD. Together, our study suggests that N17 modifies nuclear pathogenesis and disease severity in HD mice by regulating subcellular localization of known nuclear pathogenic mHTT species.
Asunto(s)
Nucléolo Celular/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Expansión de Repetición de Trinucleótido/genética , Factores de Edad , Animales , Encéfalo/metabolismo , Encéfalo/patología , Nucléolo Celular/patología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/genética , Células HEK293/ultraestructura , Humanos , Proteína Huntingtina , Enfermedad de Huntington/complicaciones , Locomoción/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Tamaño de los Órganos/genética , Fenotipo , Estructura Terciaria de Proteína/genética , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/patologíaRESUMEN
OBJECTIVE: To elucidate the clinical and cellular characteristics of spinocerebellar ataxia type 35 (SCA35), which is caused by mutations in the TGM6 gene encoding transglutaminase 6 (TG6), in a Taiwanese cohort. METHODS: Mutations in TGM6 were ascertained in 109 unrelated probands of Chinese descent with molecularly unassigned SCA from 512 pedigrees, in whom mutations responsible for 15 other ataxia syndromes had been excluded. The clinical features of all patients with a TGM6 mutation were systematically analyzed. The biological consequences of the newly identified TGM6 mutations were investigated in HEK293 cells transfected with mutant complementary DNA constructs. RESULTS: Two missense mutations (p.R111C and p.D510H) and one 3-base pair deletion (p.E574del) in TGM6 were identified. Among them, p.R111C and p.E574del were novel. The common features of SCA35 include a slowly progressive clinical course, trunk/limb ataxia, and hand tremors. The age at onset varies from adolescence to the fifth decade. Torticollis and intellectual impairment are rare manifestations. Brain MRI reveals diffuse cerebellar atrophy without involvement of the cerebral hemispheres or brainstem. The 3 mutations identified here attenuated the protein stability and catalytic activities of TG6. CONCLUSIONS: SCA35 is an uncommon ataxia syndrome, accounting for 0.6% (3/512) of SCAs among the Han-Chinese descent in Taiwan. This study broadens the mutational spectrum of SCA35 and stresses the importance of TG6 in cerebellar functions.
Asunto(s)
Enfermedad de Machado-Joseph/genética , Enfermedad de Machado-Joseph/fisiopatología , Mutación Missense/genética , Transglutaminasas/genética , Adulto , Edad de Inicio , Encéfalo/patología , Estudios de Cohortes , Retículo Endoplásmico/metabolismo , Salud de la Familia , Femenino , Células HEK293/metabolismo , Células HEK293/ultraestructura , Humanos , Discapacidad Intelectual/etiología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Taiwán , Tortícolis/etiología , Transfección , Transglutaminasas/metabolismoRESUMEN
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Asunto(s)
Señalización del Calcio , Colorantes Fluorescentes/química , Fluorometría/métodos , Proteínas Fluorescentes Verdes/química , Neuroimagen/métodos , Neuronas/química , Péptidos/química , Transmisión Sináptica , Animales , Astrocitos/química , Astrocitos/ultraestructura , Caenorhabditis elegans , Cristalografía por Rayos X , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Colorantes Fluorescentes/análisis , Genes Sintéticos , Vectores Genéticos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Células HEK293/química , Células HEK293/ultraestructura , Hipocampo/química , Hipocampo/citología , Humanos , Larva , Rayos Láser , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Neuromuscular/química , Unión Neuromuscular/ultraestructura , Neuronas/fisiología , Neuronas/ultraestructura , Neurópilo/química , Neurópilo/fisiología , Neurópilo/ultraestructura , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/fisiología , Neuronas Receptoras Olfatorias/ultraestructura , Péptidos/análisis , Péptidos/genética , Estimulación Luminosa , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Células Bipolares de la Retina/química , Células Bipolares de la Retina/fisiología , Células Bipolares de la Retina/ultraestructura , Pez Cebra/crecimiento & desarrolloRESUMEN
Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-µm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.
Asunto(s)
Células HEK293/ultraestructura , Microscopía Electrónica de Transmisión de Rastreo/métodos , Microscopía Electrónica de Rastreo/métodos , Algoritmos , Tomografía con Microscopio Electrónico/ética , Tomografía con Microscopio Electrónico/métodos , Humanos , Microscopía Electrónica de Rastreo/instrumentación , Microscopía Electrónica de Transmisión de Rastreo/instrumentaciónRESUMEN
Due to their role in producing energy, as major sources of free radicals, and as critical regulators of apoptosis, mitochondria play a dominant role in the central nervous system (CNS). Mitochondrial dysfunction represents one major pathomechanism of Alzheimer's disease (AD), including impaired function of mitochondrial respiratory chain complexes and deficits of mitochondrial dynamics, such as impaired balance between fission and fusion mechanisms and reduced mitochondrial trafficking. Major consequences are enhanced depletion of mitochondria in axons and dendrites, synaptic dysfunction, and finally neuronal loss. Interfering with impaired mitochondrial dynamics has been proposed as novel strategy for antidementia drugs. Dimebon has been shown to improve cognition in animal models and seems to be beneficial in AD patients. Regardless of the final proof of Dimebon's clinical efficacy, it might specifically interfere with mechanisms relevant for the cognitive decline, especially by improving impaired mitochondrial function and/or dynamics in AD. Herein, we tested the effects of Dimebon on mitochondrial function and dynamics in a cellular model, overexpressing neurotoxic Aß peptides, one of the hallmarks of AD. Dimebon exerted pronounced effects on mitochondrial morphology, respiratory chain complex activities, and enlarged mitochondrial mass. In summary, form and function of mitochondria are altered in the Aß overexpressing cell model and precisely those changes are restored by nanomolar Dimebon treatment. Our findings support the idea that Dimebon improves mitochondrial function and that these "disease specific" effects might be relevant for interpretation and planning of future clinical trials.