Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
1.
Nature ; 514(7522): 328-34, 2014 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-25119039

RESUMO

Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.


Assuntos
Microscopia Crioeletrônica , Receptores de AMPA/metabolismo , Receptores de AMPA/ultraestrutura , Receptores de Ácido Caínico/metabolismo , Receptores de Ácido Caínico/ultraestrutura , Animais , Sítios de Ligação , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Estrutura Terciária de Proteína/efeitos dos fármacos , Ratos , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/química , Receptores de Ácido Caínico/química , Receptor de GluK2 Cainato
2.
Appl Environ Microbiol ; 83(3)2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27836840

RESUMO

SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 µm3 Strikingly, the periplasmic space occupied ∼20% to 50% of the total cell volume in log-phase cells and ∼50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/µm3 Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. IMPORTANCE: Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of "Candidatus Pelagibacter ubique," the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria.


Assuntos
Alphaproteobacteria/ultraestrutura , Alphaproteobacteria/fisiologia , Tomografia com Microscopia Eletrônica , Água do Mar/microbiologia
3.
Mol Pharmacol ; 89(6): 645-51, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27036132

RESUMO

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.


Assuntos
Microscopia Crioeletrônica/métodos , Glutamato Desidrogenase/ultraestrutura , Animais , Bovinos , Cristalografia por Raios X , Glutamato Desidrogenase/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Ligantes , Mamíferos , Modelos Moleculares , NAD/química , NAD/metabolismo , Estrutura Quaternária de Proteína
4.
PLoS Pathog ; 6(3): e1000794, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20221442

RESUMO

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.


Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Macrófagos/microbiologia , Mycobacterium/metabolismo , Mycobacterium/ultraestrutura , Cápsulas Bacterianas/ultraestrutura , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Citocinas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia Imunoeletrônica , Mycobacterium bovis/metabolismo , Mycobacterium bovis/ultraestrutura , Mycobacterium marinum/metabolismo , Mycobacterium marinum/ultraestrutura , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/ultraestrutura , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura
5.
J Struct Biol ; 173(2): 345-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20863895

RESUMO

Vitreous cryo-section-induced compression influences the interpretation and the reliability of electron microscopy images and tomographic reconstructions. Previous studies of this deformation have been focused at the cellular level where considerable compression occurs, yet the degree of possible intracellular macromolecular deformation has remained unclear. Here, electron cryo-tomographic reconstructions of vitreous cryo-sections show that 80S ribosomes, both intracellular and in an isolated state, appear able to resist section-induced compression. Our observations indicate that vitreous section-induced compression is non-uniform between whole cells that have been sectioned and their intracellular macromolecular complexes. We conclude that electron cryo-tomography of vitreous cryo-sections, in spite of section-induced compression, is a suitable technique for charting the structural organization of cellular nanomachines, such as ribosomes, in a cellular environment.


Assuntos
Tomografia com Microscopia Eletrônica/métodos , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura
6.
J Electron Microsc (Tokyo) ; 60 Suppl 1: S93-100, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21844602

RESUMO

A major objective of modern structural biology is to appreciate the cellular organization by elucidating the spatial arrangement of macromolecular complexes within a cell. Cryogenic sample preparation, combined with cryo-ultramicrotomy, enables large cells and pieces of biological tissues to be thinned for electron cryo-tomography, which provides a three-dimensional view of the biological sample. There are, however, limitations associated with the technique that must be realized, addressed and overcome for the procedure to become mainstream. Here, we provide perspectives on the continued advancements in cryogenic sample preparation for vitreous cryo-sectioning, image collection and post-image processing that have expanded the attainable information limit within the three-dimensional reconstructions of cells and pieces of biological tissues.


Assuntos
Crioultramicrotomia/métodos , Tomografia com Microscopia Eletrônica/métodos , Animais , Microscopia Crioeletrônica/métodos , Congelamento , Processamento de Imagem Assistida por Computador/métodos
7.
Traffic ; 9(10): 1653-64, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18694438

RESUMO

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae is largely divided between perinuclear and cortical compartments. Yeast Nvj1 localizes exclusively to small patches on the perinuclear ER where it interacts with Vac8 in the vacuole membrane to form nucleus-vacuole (NV) junctions. Three regions of Nvj1 mediate the biogenesis of NV junctions. A membrane-spanning domain targets the protein to the ER. The C-terminus binds Vac8 in the vacuole membrane, which induces the clustering of both proteins into NV junctions. The luminal N-terminus is required for strict perinuclear localization. Three-dimensional cryo-electron tomography reveals that Nvj1 clamps the separation between the two nuclear membranes to half the width of bulk nuclear envelope. The N-terminus contains a hydrophobic sequence bracketed by basic residues that resembles outer mitochondrial membrane signal-anchors. The hydrophobic sequence can be scrambled or reversed without affecting function. Mutations that reduce the hydrophobicity of the core sequence or affect the distribution of basic residues cause mislocalization to the cortical ER. We conclude that the N-terminus of Nvj1 is a retention sequence that bridges the perinuclear lumen and inserts into the inner nuclear membrane.


Assuntos
Retículo Endoplasmático/metabolismo , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Microscopia Crioeletrônica , Retículo Endoplasmático/ultraestrutura , Genes Reporter , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Microscopia Confocal , Dados de Sequência Molecular , Membrana Nuclear/ultraestrutura , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Vacúolos/ultraestrutura
8.
J Struct Biol ; 169(2): 219-25, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19822214

RESUMO

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images. Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50nm, vitreous cryo-sections of Saccharomyces cerevisiae.


Assuntos
Microscopia Crioeletrônica/métodos , Crioultramicrotomia/métodos , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/instrumentação , Crioultramicrotomia/instrumentação , Tomografia com Microscopia Eletrônica/instrumentação , Umidade , Ribossomos/ultraestrutura , Saccharomyces cerevisiae , Eletricidade Estática
9.
Histochem Cell Biol ; 132(3): 253-62, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19649648

RESUMO

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be 'docked' or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as 'high pressure freezing' with 'freeze-substitution followed by room temperature plastic sectioning' or 'cryo-sectioning of unperturbed vitreous fully hydrated samples' have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported.


Assuntos
Microscopia Crioeletrônica/métodos , Nanoestruturas/ultraestrutura , Organelas/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Tomografia/métodos
10.
Sci Rep ; 4: 7084, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25403871

RESUMO

Poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits structural determination by single particle cryo-electron microscopy (cryo-EM). Here, we present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. We demonstrate the utility of this approach to drive partitioning of ionotropic glutamate receptors into the holes, thereby enabling 3D structural analysis using cryo-EM methods.


Assuntos
Proteínas de Bactérias/química , Chaperonina 60/química , Microscopia Crioeletrônica/instrumentação , Receptores de AMPA/química , Receptores de Ácido Caínico/química , Animais , Carbono/química , Ouro/química , Porosidade , Ratos , Molhabilidade , Receptor de GluK2 Cainato
11.
Neurobiol Aging ; 34(6): 1621-31, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23481568

RESUMO

During prion disease, cellular prion protein (PrP(C)) is refolded into a pathogenic isoform (PrP(Sc)) that accumulates in the central nervous system and causes neurodegeneration and death. We used immunofluorescence, quantitative cryo-immunogold EM, and tomography to detect nascent, full-length PrP(Sc) in the hippocampus of prion-infected mice from early preclinical disease stages onward. Comparison of uninfected and infected brains showed that sites containing full-length PrP(Sc) could be recognized in the neuropil by bright spots and streaks of immunofluorescence on semi-thin (200-nm) sections, and by clusters of cryo-immunogold EM labeling. PrP(Sc) was found mainly on neuronal plasma membranes, most strikingly on membrane invaginations and sites of cell-to-cell contact, and was evident by 65 days postinoculation, or 54% of the incubation period to terminal disease. Both axons and dendrites in the neuropil were affected. We hypothesize that closely apposed plasma membranes provide a favorable environment for prion conversion and intercellular prion transfer. Only a small proportion of clustered PrP immunogold labeling was found at synapses, indicating that synapses are not targeted specifically in prion disease.


Assuntos
Química Encefálica , Encéfalo/patologia , Membrana Celular/química , Membrana Celular/patologia , Proteínas PrPSc/metabolismo , Doenças Priônicas/patologia , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Comunicação Celular , Membrana Celular/ultraestrutura , Camundongos , Proteínas PrPSc/química , Proteínas PrPSc/ultraestrutura , Doenças Priônicas/metabolismo
12.
PLoS One ; 8(11): e78187, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244293

RESUMO

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Anti-HIV/química , Proteína gp41 do Envelope de HIV/química , HIV-1/química , Fragmentos Fab das Imunoglobulinas/química , Humanos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
13.
FEBS J ; 280(1): 28-45, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23181775

RESUMO

Cryo-electron microscopy (cryo-EM) is increasingly becoming a mainstream technology for studying the architecture of cells, viruses and protein assemblies at molecular resolution. Recent developments in microscope design and imaging hardware, paired with enhanced image processing and automation capabilities, are poised to further advance the effectiveness of cryo-EM methods. These developments promise to increase the speed and extent of automation, and to improve the resolutions that may be achieved, making this technology useful to determine a wide variety of biological structures. Additionally, established modalities for structure determination, such as X-ray crystallography and nuclear magnetic resonance spectroscopy, are being routinely integrated with cryo-EM density maps to achieve atomic-resolution models of complex, dynamic molecular assemblies. In this review, which is directed towards readers who are not experts in cryo-EM methodology, we provide an overview of emerging themes in the application of this technology to investigate diverse questions in biology and medicine. We discuss the ways in which these methods are being used to study structures of macromolecular assemblies that range in size from whole cells to small proteins. Finally, we include a description of how the structural information obtained by cryo-EM is deposited and archived in a publicly accessible database.


Assuntos
Microscopia Crioeletrônica/métodos , Animais , Tomografia com Microscopia Eletrônica , Análise de Fourier , Humanos , Imageamento Tridimensional , Substâncias Macromoleculares/ultraestrutura , Proteínas/ultraestrutura
14.
Mol Biol Cell ; 23(3): 423-32, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22160597

RESUMO

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.


Assuntos
Saccharomyces cerevisiae/química , Septinas/química , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Imageamento Tridimensional , Mutação , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Septinas/ultraestrutura
15.
Cell ; 129(7): 1287-98, 2007 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-17604718

RESUMO

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.


Assuntos
Citosol/fisiologia , Lisossomos/fisiologia , Mycobacterium/fisiologia , Células Mieloides/microbiologia , Fagossomos/fisiologia , Apresentação de Antígeno/fisiologia , Biomarcadores/metabolismo , Compartimento Celular/fisiologia , Morte Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Citosol/ultraestrutura , Regulação Bacteriana da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Humanos , Imuno-Histoquímica , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Lisossomos/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Mycobacterium/genética , Mycobacterium/ultraestrutura , Mycobacterium leprae/genética , Mycobacterium leprae/fisiologia , Mycobacterium leprae/ultraestrutura , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Mycobacterium tuberculosis/ultraestrutura , Células Mieloides/fisiologia , Células Mieloides/ultraestrutura , Fagossomos/ultraestrutura
16.
J Microsc ; 224(Pt 2): 129-34, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17204058

RESUMO

Sectioning vitrified cells and tissues for cryo-electron microscopy is more challenging than room-temperature sectioning of plastic-embedded samples. As the sample must be kept very cold (<-130 degrees C) and because there is no liquid upon which the sections can float as they are cut, transferring the sections from the knife edge to a grid is one of the more difficult steps in the process. We employed a micromanipulator to hold and control the cryo-sections as they come off the knife. This allows slower cutting speeds than are typically used in vitreous cryo-sectioning and contributes to better control during cutting, which facilitates repeatable placement of a ribbon of sections onto a grid. The ribbon is kept under tension during the entire cutting process, which may decrease folding and/or compression, features that are inherent to vitreous sections. Furthermore, the added control afforded by this technique makes it easier for multiple ribbons to be placed on a single grid, thereby increasing the number of sections that can be examined and imaged during a microscopy session. It even allows for serial cryo-electron microscopy. As such, this approach is an advance in the cryo-microtomy of vitreous sections.


Assuntos
Microscopia Crioeletrônica/métodos , Micromanipulação , Microtomia/métodos , Saccharomyces cerevisiae/ultraestrutura , Secções Congeladas/métodos , Microtomia/instrumentação
17.
Science ; 313(5789): 944-8, 2006 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-16917055

RESUMO

Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for the way dynein generates force to slide microtubules. They also reveal two dynein linkers that may provide "hard-wiring" to coordinate motor enzyme action, both circumferentially and along the axoneme. Periodic densities were also observed inside doublet microtubules; these may contribute to doublet stability.


Assuntos
Dineínas/química , Dineínas/ultraestrutura , Flagelos/ultraestrutura , Microtúbulos/ultraestrutura , Cauda do Espermatozoide/ultraestrutura , Animais , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Chlamydomonas reinhardtii/ultraestrutura , Microscopia Crioeletrônica , Dineínas/fisiologia , Flagelos/química , Flagelos/fisiologia , Congelamento , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Masculino , Proteínas Associadas aos Microtúbulos , Microtúbulos/química , Microtúbulos/fisiologia , Modelos Biológicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/ultraestrutura , Estrutura Terciária de Proteína , Ouriços-do-Mar , Cauda do Espermatozoide/química , Cauda do Espermatozoide/fisiologia , Tomografia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA