RESUMO
Continuing the celebration of Cell's 50th anniversary, this Focus Issue is an ode to the field of Structural Biology. We present Leading Edge articles highlighting specific approaches and insights that this field offers to answer fundamental and critical biological questions.
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Biologia Celular , Biologia Molecular , Publicações Periódicas como Assunto , Microscopia EletrônicaRESUMO
Biology spans a continuum of length and time scales. Individual experimental methods only glimpse discrete pieces of this spectrum but can be combined to construct a more holistic view. In this Review, we detail the latest advancements in volume electron microscopy (vEM) and cryo-electron tomography (cryo-ET), which together can visualize biological complexity across scales from the organization of cells in large tissues to the molecular details inside native cellular environments. In addition, we discuss emerging methodologies for integrating three-dimensional electron microscopy (3DEM) imaging with multimodal data, including fluorescence microscopy, mass spectrometry, single-particle analysis, and AI-based structure prediction. This multifaceted approach fills gaps in the biological continuum, providing functional context, spatial organization, molecular identity, and native interactions. We conclude with a perspective on incorporating diverse data into computational simulations that further bridge and extend length scales while integrating the dimension of time.
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Biologia , Microscopia Eletrônica , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Microscopia de Fluorescência , Tempo , Simulação por ComputadorRESUMO
High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.
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Drosophila melanogaster , Microscopia Eletrônica , Neurotransmissores , Sinapses , Animais , Encéfalo/ultraestrutura , Encéfalo/metabolismo , Conectoma , Drosophila melanogaster/ultraestrutura , Drosophila melanogaster/metabolismo , Ácido gama-Aminobutírico/metabolismo , Microscopia Eletrônica/métodos , Redes Neurais de Computação , Neurônios/metabolismo , Neurônios/ultraestrutura , Neurotransmissores/metabolismo , Sinapses/ultraestrutura , Sinapses/metabolismoRESUMO
Nearly all neurons contain a primary cilium, but little is known about how this compartment contributes to neuromodulatory signaling. In a new study, Sheu et al. use cutting-edge electron microscopy and fluorescence imaging techniques to reveal a new type of synapse that enables chemical transmission between serotonergic axons and the primary cilia of hippocampal neurons.
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Cílios , Neurônios/fisiologia , Sinapses , Hipocampo/citologia , Microscopia EletrônicaRESUMO
We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from â¼250 × 140 × 90 µm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.
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Neocórtex , Animais , Camundongos , Microscopia Eletrônica , Neocórtex/fisiologia , Organelas , Células Piramidais/fisiologia , Sinapses/fisiologiaRESUMO
Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.
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Diferenciação Celular/genética , Organoides/citologia , Organoides/metabolismo , Retina/citologia , Retina/metabolismo , Análise de Célula Única/métodos , Sinapses/fisiologia , Transcriptoma/genética , Técnicas de Cultura de Células/métodos , Linhagem Celular , Eletrofisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Predisposição Genética para Doença/genética , Humanos , Hibridização In Situ , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia Eletrônica , Família Multigênica , Naftoquinonas , Organoides/efeitos da radiação , Organoides/ultraestrutura , Retina/patologia , Retina/efeitos da radiaçãoRESUMO
The beauty of the eukaryotic cilium has been appreciated since electron microscopy first revealed its 9-fold symmetry. In this issue of Cell, Ma et al. use cryoelectron microscopy and modeling to define doublet microtubules at near-atomic resolution, revealing an intricate array of proteins decorating the inner and outer surfaces.
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Cílios , Microtúbulos , Microscopia Crioeletrônica , Microscopia Eletrônica , ProteínasRESUMO
Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent d-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research.
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Bactérias/metabolismo , Parede Celular/metabolismo , Peptidoglicano/biossíntese , Aminoácidos/química , Bactérias/ultraestrutura , Parede Celular/ultraestrutura , Corantes Fluorescentes/química , Microscopia de Força Atômica , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. VIDEO ABSTRACT.
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Mapeamento Encefálico/métodos , Conectoma/métodos , Rede Nervosa/anatomia & histologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/diagnóstico por imagem , Dendritos , Drosophila melanogaster/anatomia & histologia , Feminino , Microscopia Eletrônica/métodos , Corpos Pedunculados , Neurônios , Olfato/fisiologia , SoftwareRESUMO
How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.
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Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Membrana Celular/química , Cristalografia por Raios X , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/químicaRESUMO
Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.
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Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Queratinócitos/ultraestrutura , Pseudópodes/química , Pseudópodes/ultraestrutura , Animais , Membrana Celular/química , Queratinócitos/química , Microscopia Eletrônica , Peixe-ZebraRESUMO
The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.
Assuntos
Quinase 5 de Receptor Acoplado a Proteína G/química , Mamíferos/metabolismo , Receptores Adrenérgicos beta 2/química , Animais , Camelídeos Americanos , Bovinos , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Humanos , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Ratos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismoRESUMO
Annulate lamellae (AL) are stacked ER-derived membranes containing nuclear pore complex-like structures whose fate and function have remained a mystery. During the short interphase of early embryonic cells, AL are rapidly delivered into the nuclear envelope through fenestrations, highlighting the remarkable dynamics of the nuclear envelope.
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Membrana Nuclear/química , Poro Nuclear , Microscopia EletrônicaRESUMO
Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.
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Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Sobreviventes , Animais , Reações Cruzadas , Modelos Animais de Doenças , Mapeamento de Epitopos , Cobaias , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Modelos Moleculares , Mutagênese , UgandaRESUMO
Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , AMP Cíclico/metabolismo , Endossomos/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Microscopia Confocal , Microscopia Eletrônica , Complexos Multiproteicos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , beta-Arrestinas/químicaRESUMO
Type IV secretion systems (T4SSs) are large multisubunit translocons, found in both gram-negative and gram-positive bacteria and in some archaea. These systems transport a diverse array of substrates from DNA and protein-DNA complexes to proteins, and play fundamental roles in both bacterial pathogenesis and bacterial adaptation to the cellular milieu in which bacteria live. This review describes the various biochemical and structural advances made toward understanding the biogenesis, architecture, and function of T4SSs.
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Bactérias/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/ultraestrutura , Bactérias/química , Bactérias/classificação , Fator F/genética , Microscopia EletrônicaRESUMO
Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes.
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Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Escherichia coli/metabolismo , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.
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Nanoestruturas , Proteínas , Cristalografia por Raios X , Nanoestruturas/química , Proteínas/química , Proteínas/metabolismo , Microscopia Eletrônica , Reprodutibilidade dos TestesRESUMO
Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Lisossomos , Animais , Humanos , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestrutura , Homeostase , Longevidade , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Motivos de Aminoácidos , Microscopia EletrônicaRESUMO
To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.