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BACKGROUND: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. METHODS: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. RESULTS: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive ß-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. CONCLUSIONS: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.
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Señalización del Calcio , Retículo Sarcoplasmático , Femenino , Ratones , Animales , Retículo Sarcoplasmático/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Mitocondrias Cardíacas/metabolismo , Calcio/metabolismoRESUMEN
Most DNA viruses replicate in the cell nucleus, although the specific sites of virion assembly are as yet poorly defined. Electron microscopy on freeze-substituted, plastic-embedded sections of murine polyomavirus (PyV)-infected 3T3 mouse fibroblasts or mouse embryonic fibroblasts (MEFs) revealed tubular structures in the nucleus adjacent to clusters of assembled virions, with virions apparently "shed" or "budding" from their ends. Promyelocytic leukemia nuclear bodies (PML-NBs) have been suggested as possible sites for viral replication of polyomaviruses (BKV and SV40), herpes simplex virus (HSV), and adenovirus (Ad). Immunohistochemistry and FISH demonstrated co-localization of the viral T-antigen (Tag), PyV DNA, and the host DNA repair protein MRE11, adjacent to the PML-NBs. In PMLâ»/â» MEFs the co-localization of MRE11, Tag, and PyV DNA remained unchanged, suggesting that the PML protein itself was not responsible for their association. Furthermore, PyV-infected PMLâ»/â» MEFs and PMLâ»/â» mice replicated wild-type levels of infectious virus. Therefore, although the PML protein may identify sites of PyV replication, neither the observed "virus factories" nor virus assembly were dependent on PML. The ultrastructure of the tubes suggests a new model for the encapsidation of small DNA viruses.
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Núcleo Celular/virología , ADN Viral/metabolismo , Proteínas Nucleares/metabolismo , Infecciones por Polyomavirus/metabolismo , Poliomavirus/fisiología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ensamble de Virus/fisiología , Células 3T3 , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN Viral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Embrión de Mamíferos/virología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Proteína Homóloga de MRE11 , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Infecciones por Polyomavirus/genética , Proteína de la Leucemia Promielocítica , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Glutamatergic synapses are the primary site of excitatory synaptic signaling and neural communication in the cerebral cortex. Electron microscopy (EM) studies in non-human model organisms have demonstrated that glutamate synaptic activity and functioning are directly reflected in quantifiable ultrastructural features. Thus, quantitative EM analysis of glutamate synapses in ex vivo preserved human brain tissue has the potential to provide novel insight into in vivo synaptic functioning. However, factors associated with the acquisition and preservation of human brain tissue have resulted in persistent concerns regarding the potential confounding effects of antemortem and postmortem biological processes on synaptic and sub-synaptic ultrastructural features. Thus, we sought to determine how well glutamate synaptic relationships and nanoarchitecture are preserved in postmortem human dorsolateral prefrontal cortex (DLPFC), a region that substantially differs in size and architecture from model systems. Focused ion beam-scanning electron microscopy (FIB-SEM), a powerful volume EM (VEM) approach, was employed to generate high-fidelity, fine-resolution, three-dimensional (3D) micrographic datasets appropriate for quantitative analyses. Using postmortem human DLPFC with a 6-hour postmortem interval, we optimized a tissue preservation and staining workflow that generated samples of excellent ultrastructural preservation and the high-contrast staining intensity required for FIB-SEM imaging. Quantitative analysis of sub-cellular, sub-synaptic and organelle components within glutamate axo-spinous synapses revealed that ultrastructural features of synaptic function and activity were well-preserved within and across individual synapses in postmortem human brain tissue. The synaptic, sub-synaptic and organelle measures were highly consistent with findings from experimental models that are free from antemortem or postmortem effects. Further, dense reconstruction of neuropil revealed a unique, ultrastructurally-complex, spiny dendritic shaft that exhibited features characteristic of neuronal processes with heightened synaptic communication, integration and plasticity. Altogether, our findings provide a critical proof-of-concept that ex vivo VEM analysis provides a valuable and informative means to infer in vivo functioning of human brain.
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Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.
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In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The complex three-dimensional morphology fascinated researchers and was sometimes even the driving force to develop novel visualization techniques. However, the highly dynamic membrane systems of Golgi apparatus are delicate and prone to fixation artifacts. Therefore, the understanding of Golgi morphology and its function has been improved significantly with the development of better preparation methods. Nowadays, cryo-fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in amorphous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy and tomography. Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density. At this point, any further improvement of sample preparation would gain novel insights, perhaps not in terms of general morphology, but on fine structural details of this dynamic organelle.
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Microscopía por Crioelectrón , Aparato de Golgi/ultraestructura , Animales , Aparato de Golgi/metabolismo , HumanosRESUMEN
Cryo-electron microscopy is expanding its scope from macromolecules towards much larger and more complex cellular specimens such as organelles, cells and entire tissues. While isolated macromolecular specimens are typically composed of only very few different components that may be recognized by their shape, size or state of polymerization, cellular specimens combine large numbers of proteinaceous structures as well as nucleic acids and lipid arrays. Consequently, an unambiguous identification of these structures within the context of a whole cell may create a very difficult challenge. On plastic-embedded specimens, or Tokuyasu sections (Tokuyasu, 1980), epitopes that are exposed at the surface can be tagged by antibodies. However, vitrified sections have to be kept at strict cryo-conditions (below -140 °C) and therefore do not allow any post-sectioning treatment of the specimens other than data acquisition in the microscope. Hence, the labels have to be placed into the specimen before freezing. Here we report on the application of a small metal-clustering protein, metallothionein (MTH), as a clonable label capable of clustering metal atoms into a high-density particle with high spatial resolution. We tested MTH as a label for kinesin-decorated microtubules (MTs) as well as the building blocks of desmin intermediate filaments (IFs).
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Clonación Molecular , Microscopía por Crioelectrón , Metalotioneína/ultraestructura , Desmina/genética , Desmina/ultraestructura , Regulación de la Expresión Génica , Procesamiento de Imagen Asistido por Computador/métodos , Filamentos Intermedios/genética , Filamentos Intermedios/ultraestructura , Metalotioneína/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Moleculares , Orgánulos/ultraestructura , Estructura Terciaria de Proteína , Manejo de Especímenes/métodosRESUMEN
Trypanosoma brucei is a uni-cellular protist that causes African sleeping sickness. These parasites have a flagellum that is attached to the cell body and is indispensible for its motility. The flagellum consists of a canonical 9+2 axoneme and a paraflagellar rod (PFR), an intricate tripartite, fibrous structure that is connected to the axoneme. In this paper we describe results from cryo-electron tomography of unperturbed flagella. This method revealed novel structures that are likely involved in attaching the flagellum to the cell. We also show the first cryo-electron tomographic images of a basal body in situ, revealing electron dense structures inside its triplet microtubules. Sub-tomogram averaging of the PFR revealed that its distal region is organized as an orthorhombic crystal.
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Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Flagelos/ultraestructura , Trypanosoma brucei brucei/ultraestructura , Secciones por CongelaciónRESUMEN
Autophagy is markedly impaired in Alzheimer's disease (AD). Here we reveal unique autophagy dysregulation within neurons in five AD mouse models in vivo and identify its basis using a neuron-specific transgenic mRFP-eGFP-LC3 probe of autophagy and pH, multiplex confocal imaging and correlative light electron microscopy. Autolysosome acidification declines in neurons well before extracellular amyloid deposition, associated with markedly lowered vATPase activity and build-up of Aß/APP-ßCTF selectively within enlarged de-acidified autolysosomes. In more compromised yet still intact neurons, profuse Aß-positive autophagic vacuoles (AVs) pack into large membrane blebs forming flower-like perikaryal rosettes. This unique pattern, termed PANTHOS (poisonous anthos (flower)), is also present in AD brains. Additional AVs coalesce into peri-nuclear networks of membrane tubules where fibrillar ß-amyloid accumulates intraluminally. Lysosomal membrane permeabilization, cathepsin release and lysosomal cell death ensue, accompanied by microglial invasion. Quantitative analyses confirm that individual neurons exhibiting PANTHOS are the principal source of senile plaques in amyloid precursor protein AD models.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Autofagia , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Lisosomas/metabolismo , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Placa Amiloide/metabolismoRESUMEN
The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.
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Astrocitos/ultraestructura , Gránulos Citoplasmáticos/ultraestructura , Hipocampo/ultraestructura , Microtúbulos/ultraestructura , Neuronas/ultraestructura , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Axones/metabolismo , Axones/ultraestructura , Células Cultivadas , Microscopía por Crioelectrón/métodos , Gránulos Citoplasmáticos/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/metabolismo , Neuritas/metabolismo , Neuritas/ultraestructura , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/ultraestructura , RatasRESUMEN
BACKGROUND INFORMATION: Sphingomyelin is one of the major phospholipids in the cell nucleus. However, its intranuclear distribution with regard to different functional nuclear domains as well as its possible involvement in the nuclear functional architecture remains to be elucidated. RESULTS: We carried out an ultrastructural cytochemical study of the intranuclear distribution of SM (sphingomyelin) using an in situ binding assay of neutral SMase (sphingomyelinase) conjugated to colloidal gold particles. The enzymatic labelling was carried out on ultrathin sections of different mammalian cells prepared by means of various fixation and resin-embedding protocols. Transmission electron microscopic analysis revealed preferential localization of SM within the PR (perichromatin region), a functionally important nucleoplasmic domain containing sites of pre-mRNA synthesis and processing. In the nucleolus, SM is mostly associated with the dense fibrillar component containing transcriptionally active ribosomal genes. Microinjection of enzymatically active SMase into living cells resulted in a rapid degradation of intranuclear structure. CONCLUSIONS: Our observations, supported by biochemical data, provide evidence for the involvement of SM in important nuclear functions. They bring additional information pointing out the PR as an essential functional nuclear domain. Furthermore, they suggest a role for SM in the internal nuclear architecture.
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Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cromatina/metabolismo , Cromatina/ultraestructura , Esfingomielinas/metabolismo , Animales , Ratones , Microscopía Electrónica de Transmisión , Ratas , Transcripción GenéticaRESUMEN
Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.
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Células Cultivadas/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía de Sonda de Barrido/métodos , Microscopía/métodos , Neoplasias Óseas/patología , Línea Celular Tumoral , Marcadores Fiduciales , Colorantes Fluorescentes , Oro , Humanos , Microscopía Electrónica de Rastreo , Mitocondrias/ultraestructura , Nanotubos , Osteosarcoma/patología , Flujo de TrabajoRESUMEN
The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (µCT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. µCT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.
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Imagenología Tridimensional , Válvula Pulmonar , Animales , Ratones , Microscopía Electrónica de Rastreo , Válvula Pulmonar/diagnóstico por imagen , Válvula Pulmonar/cirugía , Manejo de Especímenes , Microtomografía por Rayos XRESUMEN
The solid-electrolyte interphase (SEI), a layer formed on the electrode surface, is essential for electrochemical reactions in batteries and critically governs the battery stability. Active materials, especially those with extremely high energy density, such as silicon (Si), often inevitably undergo a large volume swing upon ion insertion and extraction, raising a critical question as to how the SEI interactively responds to and evolves with the material and consequently controls the cycling stability of the battery. Here, by integrating sensitive elemental tomography, an advanced algorithm and cryogenic scanning transmission electron microscopy, we unveil, in three dimensions, a correlated structural and chemical evolution of Si and SEI. Corroborated with a chemomechanical model, we demonstrate progressive electrolyte permeation and SEI growth along the percolation channel of the nanovoids due to vacancy injection and condensation during the delithiation process. Consequently, the Si-SEI spatial configuration evolves from the classic 'core-shell' structure in the first few cycles to a 'plum-pudding' structure following extended cycling, featuring the engulfing of Si domains by the SEI, which leads to the disruption of electron conduction pathways and formation of dead Si, contributing to capacity loss. The spatially coupled interactive evolution model of SEI and active materials, in principle, applies to a broad class of high-capacity electrode materials, leading to a critical insight for remedying the fading of high-capacity electrodes.
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While fluorescence microscopy provides tools for highly specific labeling and sensitive detection, its resolution limit and lack of general contrast has hindered studies of cellular structure and protein localization. Recent advances in correlative light and electron microscopy (CLEM), including the fully integrated CLEM workflow instrument, the FEI CorrSight with MAPS, have allowed for a more reliable, reproducible, and quicker approach to correlate three-dimensional time-lapse confocal fluorescence data, with three-dimensional focused ion beam-scanning electron microscopy data. Here we demonstrate the entire integrated CLEM workflow using fluorescently tagged MCF7 breast cancer cells.
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Imagenología Tridimensional , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células MCF-7 , Microfluídica , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility. METHODOLOGY/PRINCIPAL FINDINGS: We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum. CONCLUSIONS/SIGNIFICANCE: The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.
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Flagelos/ultraestructura , Trypanosoma brucei brucei/ultraestructura , Axonema/metabolismo , Axonema/ultraestructura , Microscopía por Crioelectrón , Flagelos/metabolismo , Humanos , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/parasitologíaRESUMEN
Traditionally, cellular specimens and tissues have been prepared for electron microscopy by chemical fixation or rapid freezing followed by freeze substitution and plastic embedding. Once embedded in a solid polymer, sections are obtained by room-temperature microtomy. More recently, two developments added a new twist to cellular electron microscopy: first, the rediscovery of tomographic 3-D reconstruction methods, which do not depend on any type of averaging procedures and hence are well suited for large irregular structures such as cellular organelles and large-macromolecular assemblies. Second, new advances made for the preparation of sectioned vitrified cellular specimens enable imaging them not only after plastic embedding but also directly in their vitrified state, recorded under strict cryo- and low-dose conditions. Today, tomography strongly benefits from an ever-evolving computer environment that now allows handling very large datasets with reasonable speed. Cellular tomography can be divided into four different classes: (A) tomography on thick (~300 nm) sections prepared from plastic embedded chemically fixed or freeze-substituted cells and tissues. (B) Large-scale tomographic 3-D data of entire cells obtained by serial sectioning of plastic embedded chemically fixed or freeze-substituted specimens obtained by computationally merging tomographic data from many consecutive sections within the grid plane and also in Z-direction. (C) Cryo-electron microscopy (cryo-EM) techniques now allow recording tomograms of intact vitrified small cells (e.g., bacteria) without substitution or sectioning. (D) Cryo-EM and cryo-tomography are increasingly applied to thin vitrified (<100 nm) obtained under strict cryo-conditions, including the during data recording in the microscope. Without extending to molecular tomography, which is described elsewhere in this book, this chapter leads the reader through cellular tomography by following the specifications outlined above.
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Células/ultraestructura , Microscopía por Crioelectrón , Animales , Células/citología , HumanosRESUMEN
The technology to produce cryo-electron tomography on vitrified sections is now a few years old and some specialised labs worldwide have gathered sufficient experience so that it is justified at this point to critically analyse its usefulness for cellular and molecular biology, and make predictions on how the method might develop from here. Remarkably, the production of vitrified sections has been introduced some 40 years ago (the very origin dates back to Christensen, 1971, and McDowall et al., 1983). However, the real breakthrough came between 2002 and 2004 when the groups of Jacques Dubochet and Carmen Manella independently resurrected the vitrified sectioning technology from its sleeping beauty state. And despite its hooks and hurdles a beauty indeed it is! When aiming at the right subjects the results obtained by vitrified sectioning and soon after by cryo-electron tomography exceeded all expectations. Molecular details of intracellular structures were imaged with never before seen clarity in a comparable setting, and the structural preservation of macromolecular assemblies within cells was stunning. However, as with every progress, the great results we now have with vitrified sectioning come at a price. The sectioning procedure and handling of vitrified sections is tricky and requires substantial training and experience. Once frozen, the specimens cannot be manipulated anymore (e.g., by staining or immuno-labelling). The contrast, as with all true cryo-EM approaches, is produced solely by small density differences between cytosol and macromolecular assemblies, membranes, or nucleic acid structures (e.g., ribosomes, nucleosomes, inner nuclear structures, etc.). Vitrified sectioning should not be seen as a competition to the more established plastic-section tomography, but constitutes an excellent complement, filling in high-resolution detail in the overview of cellular architecture. Here we critically compare the benefits and limitations of vitrified sectioning for its application to modern structural cell biology.
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Biología Celular/instrumentación , Tomografía con Microscopio Electrónico , Secciones por Congelación , Animales , Artefactos , Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Together with microtubules and actin filaments (F-actin), intermediate filaments (IFs) form the cytoskeleton of metazoan cells. However, unlike the other two entities that are extremely conserved, IFs are much more diverse and are grouped into five different families. In contrast to microtubules and F-actin, IFs do not exhibit a polarity, which may be the reason that no molecular motors travel along them. The molecular structure of IFs is less well resolved than that of the other cytoskeletal systems. This is partially due to their functional variability, tissue-specific expression, and their intrinsic structural properties. IFs are composed mostly of relatively smooth protofibrils formed by antiparallel arranged α-helical coiled-coil bundles flanked by small globular domains at either end. These features make them difficult to study by various electron microscopy methods or atomic force microscopy (AFM). Furthermore, the elongated shape of monomeric or dimeric IF units interferes with the formation of highly ordered three-dimensional (3-D) crystals suitable for atomic resolution crystallography. So far, most of the data we currently have on IF macromolecular structures come from electron microscopy of negatively stained samples, and fragmented α-helical coiled-coil units solved by X-ray diffraction. In addition, AFM allows the observation of the dynamic states of IFs in solution and delivers a new view into the assembly properties of IFs. Here, we discuss the applicability of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for the field. Both methods are strongly related and have only recently been applied to IFs. However, cryo-EM revealed distinct new features within IFs that have not been seen before, and cryo-ET adds a 3-D view of IFs revealing the path and number of protofilaments within the various IF assemblies.
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Microscopía por Crioelectrón/métodos , Imagenología Tridimensional/métodos , Filamentos Intermedios/ultraestructura , Animales , Microscopía por Crioelectrón/instrumentación , Criopreservación/métodos , Desmina/ultraestructura , Tomografía con Microscopio Electrónico/instrumentación , Tomografía con Microscopio Electrónico/métodos , Imagenología Tridimensional/instrumentación , Filamentos Intermedios/química , Microscopía de Fuerza Atómica/instrumentación , Microscopía de Fuerza Atómica/métodos , Coloración Negativa/métodos , Vimentina/ultraestructuraRESUMEN
Nuclear architecture has been investigated intensively by various electron microscopy (EM) methods. Most of these require chemical fixation of the sample, although cryofixation has also been used in combination with cryosubstitution and resin embedding. This approach allowed one to considerably increase the knowledge about the structural features of different nuclear domains and their involvement in nuclear functions. Cryoelectron microscopy of vitreous sections (CEMOVIS) has added a new dimension to the ultrastructural analysis of the cell nucleus, especially thanks to the possibility of observing the specimen in its hydrated state. In this way one can analyse, at high resolution, cellular structures as close as possible to their native state. In this chapter we describe in detail the different steps of the CEMOVIS method, which should allow an electron microscopist to perform cryosectioning and cryoelectron microscopy of vitrified biological material.
Asunto(s)
Núcleo Celular/ultraestructura , Microscopía por Crioelectrón/métodos , Animales , Línea Celular Tumoral , Crioultramicrotomía , RatasRESUMEN
Neurospora crassa kinesin NcKin3 belongs to a unique fungal-specific subgroup of small Kinesin-3-related motor proteins. One of its functions appears to be the transport of mitochondria along microtubules. Here, we present the X-ray structure of a C-terminally truncated monomeric construct of NcKin3 comprising the motor domain and the neck linker, and a 3-D image reconstruction of this motor domain bound to microtubules, by cryoelectron microscopy. The protein contains Mg.ADP bound to the active site, yet the structure resembles an ATP-bound state. By comparison with structures of the Kinesin-3 motor Kif1A in different nucleotide states (Kikkawa, M. et al. (2001) Nature (London, U.K.) 411, 439-445), the NcKin3 structure corresponds to the AMPPCP complex of Kif1A rather than the AMPPNP complex. NcKin3-specific differences in the coordination of the nucleotide and asymmetric interactions between adjacent molecules in the crystal are discussed in the context of the unusual kinetics of the dimeric wild-type motor and the monomeric construct used for crystal structure analysis. The NcKin3 motor decorates microtubules at a stoichiometry of one head per alphabeta-tubulin heterodimer, thereby forming an axial periodicity of 8 nm. In spite of unusual extensions at the N-terminus and within flexible loops L2, L8a, and L12 (corresponding to the K-loop of monomeric kinesins), the microtubule binding geometry is similar to that of other members of the kinesin family.