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1.
Cell ; 186(9): 1877-1894.e27, 2023 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-37116470

RESUMEN

Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.


Asunto(s)
Virus de la Parotiditis , Infección Persistente , Humanos , Virus de la Parotiditis/fisiología , Nucleocápside , Fosfoproteínas/metabolismo , Replicación Viral
2.
Cell ; 185(8): 1308-1324.e23, 2022 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-35325593

RESUMEN

Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Animales , Gránulos de Ribonucleoproteínas Citoplasmáticas , Drosophila/embriología , Proteínas de Drosophila/genética , Desarrollo Embrionario , Oocitos/metabolismo , ARN/metabolismo
3.
Cell ; 181(2): 346-361.e17, 2020 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-32302572

RESUMEN

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformación de Ácido Nucleico , Orgánulos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Estrés Fisiológico/genética
4.
Cell ; 169(6): 1066-1077.e10, 2017 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-28575670

RESUMEN

Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin ∼4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrosoma/química , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Caenorhabditis elegans/citología , Proteínas Portadoras/metabolismo , Centrosoma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo
5.
Mol Cell ; 84(14): 2698-2716.e9, 2024 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-39059370

RESUMEN

The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.


Asunto(s)
Citoplasma , Polirribosomas , Ribonucleoproteínas , Polirribosomas/metabolismo , Citoplasma/metabolismo , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Simulación de Dinámica Molecular , ARN Mensajero/metabolismo , ARN Mensajero/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Condensados Biomoleculares/metabolismo , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética
6.
Cell ; 166(6): 1572-1584.e16, 2016 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-27594427

RESUMEN

P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory, we show that, in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development by using RNA competition to regulate local phase separation.


Asunto(s)
Caenorhabditis elegans/metabolismo , ARN Mensajero/metabolismo , Animales , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/metabolismo , Polaridad Celular , Embrión no Mamífero , Espacio Intracelular/química , Espacio Intracelular/metabolismo , Modelos Teóricos , Unión Proteica , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo
7.
Cell ; 162(5): 1066-77, 2015 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-26317470

RESUMEN

Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.


Asunto(s)
Envejecimiento/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Mutación , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Envejecimiento/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Núcleo Celular/química , Citoplasma/química , Humanos , Priones/química , Agregado de Proteínas , Estructura Terciaria de Proteína , Proteína FUS de Unión a ARN/metabolismo
8.
Nature ; 628(8006): 47-56, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38570716

RESUMEN

Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.


Asunto(s)
Biología Celular , Células , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Microscopía por Crioelectrón/métodos , Microscopía por Crioelectrón/tendencias , Tomografía con Microscopio Electrónico/métodos , Tomografía con Microscopio Electrónico/tendencias , Sustancias Macromoleculares/análisis , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Biología Celular/instrumentación , Células/química , Células/citología , Células/metabolismo , Células/ultraestructura , Humanos
9.
Nature ; 610(7930): 205-211, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36171285

RESUMEN

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells1. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes4. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.


Asunto(s)
Microscopía por Crioelectrón , Mycoplasma pneumoniae , Biosíntesis de Proteínas , Proteínas Ribosómicas , Ribosomas , Antibacterianos/farmacología , Mycoplasma pneumoniae/citología , Mycoplasma pneumoniae/efectos de los fármacos , Mycoplasma pneumoniae/metabolismo , Mycoplasma pneumoniae/ultraestructura , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Polirribosomas/efectos de los fármacos , Polirribosomas/metabolismo , Polirribosomas/ultraestructura , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/ultraestructura
10.
Nat Methods ; 20(12): 1900-1908, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37932397

RESUMEN

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.


Asunto(s)
Redes Neurales de la Computación , Orgánulos , Humanos , Microscopía por Crioelectrón/métodos , Ligandos , Orgánulos/ultraestructura , Tomografía con Microscopio Electrónico/métodos
11.
Nat Methods ; 20(2): 284-294, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36690741

RESUMEN

Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (deep picker in context), an open-source deep-learning framework for supervised segmentation and macromolecular complex localization in cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles, and cytosol. By comparing DeePiCt to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, applying pre-trained networks to a HeLa cell tomogram demonstrates that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate use by the community.


Asunto(s)
Mitocondrias , Ribosomas , Humanos , Células HeLa , Tomografía con Microscopio Electrónico/métodos , Retículo Endoplásmico , Procesamiento de Imagen Asistido por Computador/métodos
12.
Nature ; 586(7831): 796-800, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32879490

RESUMEN

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.


Asunto(s)
Microscopía por Crioelectrón , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/ultraestructura , Autofagia , Modelos Moleculares , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tomografía
13.
Proc Natl Acad Sci U S A ; 120(15): e2213149120, 2023 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-37027429

RESUMEN

Cryoelectron tomography directly visualizes heterogeneous macromolecular structures in their native and complex cellular environments. However, existing computer-assisted structure sorting approaches are low throughput or inherently limited due to their dependency on available templates and manual labels. Here, we introduce a high-throughput template-and-label-free deep learning approach, Deep Iterative Subtomogram Clustering Approach (DISCA), that automatically detects subsets of homogeneous structures by learning and modeling 3D structural features and their distributions. Evaluation on five experimental cryo-ET datasets shows that an unsupervised deep learning based method can detect diverse structures with a wide range of molecular sizes. This unsupervised detection paves the way for systematic unbiased recognition of macromolecular complexes in situ.


Asunto(s)
Tomografía con Microscopio Electrónico , Procesamiento de Imagen Asistido por Computador , Procesamiento de Imagen Asistido por Computador/métodos , Análisis por Conglomerados , Estructura Molecular , Tomografía con Microscopio Electrónico/métodos , Sustancias Macromoleculares/química , Microscopía por Crioelectrón/métodos
14.
J Struct Biol ; 216(2): 108067, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38367824

RESUMEN

Cellular cryo-electron tomography (cryo-ET) has emerged as a key method to unravel the spatial and structural complexity of cells in their near-native state at unprecedented molecular resolution. To enable quantitative analysis of the complex shapes and morphologies of lipid membranes, the noisy three-dimensional (3D) volumes must be segmented. Despite recent advances, this task often requires considerable user intervention to curate the resulting segmentations. Here, we present ColabSeg, a Python-based tool for processing, visualizing, editing, and fitting membrane segmentations from cryo-ET data for downstream analysis. ColabSeg makes many well-established algorithms for point-cloud processing easily available to the broad community of structural biologists for applications in cryo-ET through its graphical user interface (GUI). We demonstrate the usefulness of the tool with a range of use cases and biological examples. Finally, for a large Mycoplasma pneumoniae dataset of 50 tomograms, we show how ColabSeg enables high-throughput membrane segmentation, which can be used as valuable training data for fully automated convolutional neural network (CNN)-based segmentation.


Asunto(s)
Algoritmos , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Programas Informáticos , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Redes Neurales de la Computación , Membrana Celular/ultraestructura , Mycoplasma pneumoniae/ultraestructura , Interfaz Usuario-Computador , Imagenología Tridimensional/métodos
15.
Nat Methods ; 18(2): 186-193, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33542511

RESUMEN

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and inside cells. In addition to aligning individual particles, accurate registration of sample motion and three-dimensional deformation during exposures are crucial for achieving high-resolution reconstructions. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and couples a comprehensive spatial deformation model to in silico correction of electron-optical aberrations. M provides a unified optimization framework for both frame-series and tomographic tilt-series data. We show that tilt-series data can provide the same resolution as frame-series data on a purified protein specimen, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M resolves to residue level a 70S ribosome bound to an antibiotic inside intact bacterial cells. Our work provides a computational tool that facilitates structural biology in cells.


Asunto(s)
Antibacterianos/metabolismo , Microscopía por Crioelectrón/métodos , Ribosomas/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Interfaz Usuario-Computador
16.
Proc Natl Acad Sci U S A ; 118(46)2021 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-34772804

RESUMEN

Unicellular marine microalgae are responsible for one of the largest carbon sinks on Earth. This is in part due to intracellular formation of calcium carbonate scales termed coccoliths. Traditionally, the influence of changing environmental conditions on this process has been estimated using poorly constrained analogies to crystallization mechanisms in bulk solution, yielding ambiguous predictions. Here, we elucidated the intracellular nanoscale environment of coccolith formation in the model species Pleurochrysis carterae using cryoelectron tomography. By visualizing cells at various stages of the crystallization process, we reconstructed a timeline of coccolith development. The three-dimensional data portray the native-state structural details of coccolith formation, uncovering the crystallization mechanism, and how it is spatially and temporally controlled. Most strikingly, the developing crystals are only tens of nanometers away from delimiting membranes, resulting in a highly confined volume for crystal growth. We calculate that the number of soluble ions that can be found in such a minute volume at any given time point is less than the number needed to allow the growth of a single atomic layer of the crystal and that the uptake of single protons can markedly affect nominal pH values. In such extreme confinement, the crystallization process is expected to depend primarily on the regulation of ion fluxes by the living cell, and nominal ion concentrations, such as pH, become the result, rather than a driver, of the crystallization process. These findings call for a new perspective on coccolith formation that does not rely exclusively on solution chemistry.


Asunto(s)
Carbonato de Calcio/metabolismo , Microalgas/metabolismo , Cristalización/métodos , Planeta Tierra , Haptophyta/metabolismo , Concentración de Iones de Hidrógeno , Protones
17.
Nat Methods ; 17(1): 50-54, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31740821

RESUMEN

Spatially controlled cell adhesion on electron microscopy supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of electron microscopy grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex micropatterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell three-dimensional structural characterization of the underlying molecular machinery.


Asunto(s)
Membrana Celular/ultraestructura , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Imagen Molecular/métodos , Manejo de Especímenes/métodos , Membrana Celular/metabolismo , Células HeLa , Humanos , Reconocimiento de Normas Patrones Automatizadas
19.
Chromosoma ; 130(2-3): 91-102, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34091761

RESUMEN

"Interphase epichromatin" describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of "exposed" acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed "unit threads," frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with ~ 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a "sandwich" around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a "close-to-native" situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of ~ 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated ~ 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.


Asunto(s)
Cromatina , Nucleosomas , Cromatina/metabolismo , Microscopía por Crioelectrón , Interfase , Membrana Nuclear/metabolismo , Nucleosomas/metabolismo
20.
Nat Methods ; 16(8): 757-762, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31363205

RESUMEN

Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.


Asunto(s)
Caenorhabditis elegans/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Sustancias Macromoleculares/ultraestructura , Subunidades Ribosómicas/ultraestructura , Animales
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