RESUMEN
Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post-translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell-content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid-dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule-based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT.
Asunto(s)
Vesículas Extracelulares , Cinesinas , Cinesinas/metabolismo , Chaperonina con TCP-1/metabolismo , Vesículas Extracelulares/metabolismo , Metabolismo de los Lípidos , LípidosRESUMEN
We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe the approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt-series alignments, beam-induced motions of the particles throughout the tilt-series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, particularly for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.
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Electrones , Procesamiento de Imagen Asistido por Computador , Procesamiento de Imagen Asistido por Computador/métodos , Teorema de Bayes , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
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Microscopía por Crioelectrón , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestructura , Glicoproteína de la Espiga del Coronavirus/análisis , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Virión/química , Virión/ultraestructura , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Línea Celular Tumoral , Humanos , Modelos Moleculares , Docilidad , Conformación Proteica , Multimerización de Proteína , SARS-CoV-2/química , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación , Virión/aislamiento & purificación , Virión/metabolismoRESUMEN
Image-processing software has always been an integral part of structure determination by cryogenic electron microscopy (cryo-EM). Recent advances in hardware and software are recognized as one of the key factors in the so-called cryo-EM resolution revolution. Increasing computational power has opened many possibilities to consider more demanding algorithms, which in turn allow more complex biological problems to be tackled. Moreover, data processing has become more accessible to many experimental groups, with computations that used to last for many days at supercomputing facilities now being performed in hours on personal workstations. All of these advances, together with the rapid expansion of the community, continue to pose challenges and new demands on the software-development side. In this article, the development of emcore and emvis, two basic software libraries for image manipulation and data visualization in cryo-EM, is presented. The main goal is to provide basic functionality organized in modular components that other developers can reuse to implement new algorithms or build graphical applications. An additional aim is to showcase the importance of following established practices in software engineering, with the hope that this could be a first step towards a more standardized way of developing and distributing software in the field.
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Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Algoritmos , Interfaz Usuario-ComputadorRESUMEN
Cryo-electron tomography and small-angle X-ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen-hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of ~ 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are ~ 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X-ray scattering of whole chromosomes shows a main scattering peak at ~ 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis.
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Cromatina/ultraestructura , Estructuras Cromosómicas/ultraestructura , Cromosomas Humanos/ultraestructura , Metafase , Nucleosomas/ultraestructura , Tomografía con Microscopio Electrónico , Secciones por Congelación , Células HeLa , HumanosRESUMEN
MOTIVATION: Cryo electron microscopy (EM) is currently one of the main tools to reveal the structural information of biological macromolecules. The re-construction of three-dimensional (3D) maps is typically carried out following an iterative process that requires an initial estimation of the 3D map to be refined in subsequent steps. Therefore, its determination is key in the quality of the final results, and there are cases in which it is still an open issue in single particle analysis (SPA). Small angle X-ray scattering (SAXS) is a well-known technique applied to structural biology. It is useful from small nanostructures up to macromolecular ensembles for its ability to obtain low resolution information of the biological sample measuring its X-ray scattering curve. These curves, together with further analysis, are able to yield information on the sizes, shapes and structures of the analyzed particles. RESULTS: In this paper, we show how the low resolution structural information revealed by SAXS is very useful for the validation of EM initial 3D models in SPA, helping the following refinement process to obtain more accurate 3D structures. For this purpose, we approximate the initial map by pseudo-atoms and predict the SAXS curve expected for this pseudo-atomic structure. The match between the predicted and experimental SAXS curves is considered as a good sign of the correctness of the EM initial map. AVAILABILITY AND IMPLEMENTATION: The algorithm is freely available as part of the Scipion 1.2 software at http://scipion.i2pc.es/.
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Microscopía por Crioelectrón , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Rayos XRESUMEN
Since the beginning of electron microscopy, resolution has been a critical parameter. In this article, we propose a fully automatic, accurate method for determining the local resolution of a 3D map (MonoRes). The foundation of this algorithm is an extension of the concept of analytic signal, termed monogenic signal. The map is filtered at different frequencies and the amplitude of the monogenic signal is calculated, after which a criterion is applied to determine the resolution at each voxel. MonoRes is fully automatic without compulsory user parameters, with great accuracy in all tests, and is computationally more rapid than existing methods in the field. In addition, MonoRes offers the option of local filtering of the original map based on the calculated local resolution.
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Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Modelos Moleculares , Algoritmos , Simulación por Computador , Programas InformáticosRESUMEN
We have developed a new data collection method and processing framework in full field cryo soft X-ray tomography to computationally extend the depth of field (DOF) of a Fresnel zone plate lens. Structural features of 3D-reconstructed eukaryotic cells that are affected by DOF artifacts in standard reconstruction are now recovered. This approach, based on focal series projections, is easily applicable with closed expressions to select specific data acquisition parameters.
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Imagenología Tridimensional/métodos , Tomografía por Rayos X/métodos , Algoritmos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Full field soft X-ray microscopy is becoming a powerful imaging technique to analyze whole cells preserved under cryo conditions. Images obtained in these X-ray microscopes can be combined by tomographic reconstruction to quantitatively estimate the three-dimensional (3D) distribution of absorption coefficients inside the cell. The impulse response of an imaging system is one of the factors that limits the quality of the X-ray microscope reconstructions. The main goal of this work is to experimentally measure the 3D impulse response and to assess the optical resolution and depth of field of the Mistral microscope at ALBA synchrotron (Barcelona, Spain). To this end we measure the microscope apparent transfer function (ATF) and we use it to design a deblurring Wiener filter, obtaining an increase in the image quality when applied to experimental datasets collected at ALBA.
RESUMEN
We used soft X-ray three-dimensional imaging to quantify the mass of superparamagnetic iron oxide nanoparticles (SPION) within whole cells, by exploiting the iron oxide differential absorption contrast. Near-edge absorption soft X-ray nanotomography (NEASXT) combines whole-cell 3D structure determination at 50 nm resolution, with 3D elemental mapping and high throughput. We detected three-dimensional distribution of SPIONs within cells with 0.3 g/cm(3) sensitivity, sufficient for detecting the density corresponding to a single nanoparticle.
Asunto(s)
Nanopartículas del Metal , Microtomografía por Rayos X/métodos , Humanos , Células MCF-7RESUMEN
Soft X-ray tomography (SXT) is becoming a powerful imaging technique to analyze eukaryotic whole cells close to their native state. Central to the analysis of the quality of SXT 3D reconstruction is the estimation of the spatial resolution and Depth of Field of the X-ray microscope. In turn, the characterization of the Modulation Transfer Function (MTF) of the optical system is key to calculate both parameters. Consequently, in this work we introduce a fully automated technique to accurately estimate the transfer function of such an optical system. Our proposal is based on the preprocessing of the experimental images to obtain an estimate of the input pattern, followed by the analysis in Fourier space of multiple orders of a Siemens Star test sample, extending in this way its measured frequency range.
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Soft X-ray Tomographic (TomoX) microscopy has become a reality in the last years. The resolution range of this technique nicely fits between confocal and electron microscopies and will play a key role in the elucidation of the organization between the molecular and the organelle levels. In fact, it offers the possibility of imaging three-dimensional structures of hydrated biological specimens near their native state without chemical pre-treatment. Ideally, TomoX reconstructs the specimen absorption coefficients from projections of this specimen, but, unfortunately, X-ray micrographs are only an approximation to projections of the specimen, resulting in inaccuracies if a tomographic reconstruction is performed without explicitly incorporating these approximations. In an attempt to mitigate some of these inaccuracies, we develop in this work an image formation model within the approximation of assuming incoherent illumination.
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Imagenología Tridimensional/métodos , Microtomografía por Rayos X/métodos , Candida albicans/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Modelos Teóricos , Fantasmas de ImagenRESUMEN
The speed of most parallel-aligned liquid-crystal-on-silicon (LCoS) spatial light modulators (SLMs) is limited to the video rate of their drivers, which is a limitation for high-speed applications. However, the LCoS SLM presented here has a driver allowing a frequency range of up to 1011 Hz. Using the static phase modulation characterization and the static lookup table (LUT), the phase modulation characterization versus frequency shows that the SLM can operate at around 130 Hz or even higher for small phase changes and at 32 Hz for extreme phase changes. A dynamic calibration is carried out, and we propose a method allowing an increase of the frame rate while maintaining a maximum phase modulation of 2pi. Experimental results of dynamic diffractive optical elements displayed on the SLM at a frame rate of 205 Hz show that the dynamic LUT improves the reconstruction quality.
RESUMEN
The inherent distortion of a reflective parallel aligned spatial light modulator (SLM) may need compensation not only for the backplane curvature but also for other possible nonuniformities caused by thickness variations of the liquid crystal layer across the aperture. First, we build a global look-up table (LUT) of phase modulation versus the addressed gray level for the whole device aperture. Second, when a lack of spatial uniformity is observed, we define a grid of cells onto the SLM aperture and develop a multipoint calibration. The relative phase variations between neighboring cells for a uniform gray level lead us to build a multi-LUT for improved compensation. Multipoint calibration can be done using either phase-shift interferometry or Fourier diffraction pattern analysis of binary phase gratings. Experimental results show the compensation progress in diffractive optical elements displayed on two SLMs.
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Two proposals to compensate chromatic aberration of a programmable phase Fresnel lens displayed on a liquid crystal device and working under polychromatic illumination are presented. They are based on multiplexing a set of lenses, designed with a common focal length for different wavelengths, and a multicolor filter that makes each sublens work almost monochromatically. One proposal uses spatial multiplexing with mosaic aperture. The other uses a rotating scheme, a color filter against an array of lens sectors, and hybrid spatial-time integration. The central order focalization has a unique location at the focal plane. We have drastically reduced the transversal chromatic aberration of the polychromatic point spread function by properly adjusting the pupil size of each sublens. Depth of focus curves have been made coincident too for the selected wavelengths.
RESUMEN
A proposal to dynamically compensate chromatic aberration of a programmable phase Fresnel lens displayed on a liquid crystal device and working under broadband illumination is presented. It is based on time multiplexing a set of lenses, designed with a common focal length for different wavelengths, and a tunable spectral filter that makes each sublens work almost monochromatically. Both the tunable filter and the sublens displayed by the spatial light modulator are synchronized. The whole set of sublenses are displayed within the integration time of the sensor. As a result the central order focalization has a unique location at the focal plane and it is common for all selected wavelengths. Transversal chromatic aberration of the polychromatic point spread function is reduced by properly adjusting the pupil size of each sublens. Longitudinal chromatic aberration is compensated by making depth of focus curves coincident for the selected wavelengths. Experimental results are in very good agreement with theory.
RESUMEN
In color pattern recognition, color channels are normally processed separately and afterward the correlation outputs are combined. This is the definition of multichannel processing. We combine a single-channel method with nonlinear filtering based on nonlinear correlations. These nonlinear correlations yield better discrimination than common matched filtering. The method codes color information as amplitude and phase distributions and is followed by correlations related to binary decompositions. The technique is based on binary decompositions of the red, green, and blue and the hue, saturation, and intensity monochromatic channels of the reference and of the input scene, after which the binary information on the red, green, and blue channels and that of the hue, saturation, and intensity channels are encoded as different angles of a phase distribution. We have applied the method to images degraded by high levels of substitutive noise. Results show that the sliced orthogonal nonlinear generalized correlation detects the target with a high degree of discrimination when other methods fail.