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1.
Proc Natl Acad Sci U S A ; 117(14): 8032-8043, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193339

RESUMO

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.


Assuntos
Ehrlichia chaffeensis/metabolismo , Ehrlichiose/patologia , Corpos de Inclusão/metabolismo , Fosfatidilcolinas/metabolismo , Vacúolos/microbiologia , Animais , Autofagossomos/metabolismo , Membrana Celular/metabolismo , Cães , Ehrlichia chaffeensis/citologia , Ehrlichia chaffeensis/patogenicidade , Ehrlichiose/sangue , Ehrlichiose/microbiologia , Endossomos/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão/ultraestrutura , Microscopia Intravital , Microscopia Eletrônica de Varredura , Células THP-1 , Imagem com Lapso de Tempo , Vacúolos/ultraestrutura
2.
J Acoust Soc Am ; 145(4): 2601, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31046349

RESUMO

Analytical solutions are presented for broadband sound fields in three rectangular enclosures with absorption applied on the floor and ceiling, rigid sidewalls, and a vertically oriented dipole source. The solutions are intended to serve as benchmarks that can be used to assess the performance of broadband techniques, particularly energy-based methods, in a relatively straightforward configuration with precisely specified boundary conditions. A broadband Helmholtz solution is developed using a frequency-by-frequency modal approach to determine the exact band averaged mean-square pressures along spatial trajectories within each enclosure. Due to the specific choice of enclosure configuration and absorption distribution, an approximate specular solution can be obtained through a summation of uncorrelated image sources. Comparisons between the band averaged Helmholtz solution and the uncorrelated image solution reveal excellent agreement for a wide range of absorption levels and improve the understanding of correlation effects in broadband sound fields. A boundary element solution with diffuse boundaries is also presented, which produces consistently higher mean-square pressures in comparison with the specular solution, emphasizing the careful attention that must be placed on correctly modeling reflecting boundary conditions and demonstrating the errors that can result from assuming a Lambertian surface.

3.
J Biocommun ; 43(1): e4, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-36407794

RESUMO

There are currently no existing guidelines for optimizing user experience in online image databases. Here we use established usability design principles to develop a set of 50 recommendations for improving database organization, navigability, and search functionality. We conduct a resource audit of commercial and non-profit image galleries and evaluate them upon our criteria. Our findings are summarized within a basic wireframe to be used as reference for prospective image databases in the future.

4.
J Virol ; 88(18): 10327-39, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24965444

RESUMO

UNLABELLED: HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4(+) T cells and either uninfected human CD4(+) T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread. IMPORTANCE: Our findings lead to new insights into unique aspects of HIV transmission in the brain and at T cell-T cell synapses, which are thought to be a predominant mode of rapid HIV transmission early in the infection process.


Assuntos
Membrana Celular/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Sinapses/virologia , Astrócitos/ultraestrutura , Astrócitos/virologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Membrana Celular/ultraestrutura , Infecções por HIV/transmissão , Humanos , Imageamento Tridimensional , Microscopia Eletrônica de Transmissão , Sinapses/ultraestrutura
5.
Proc Natl Acad Sci U S A ; 110(12): 4592-7, 2013 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-23460696

RESUMO

Rapid antigenic variation of HA, the major virion surface protein of influenza A virus, remains the principal challenge to the development of broader and more effective vaccines. Some regions of HA, such as the stem region proximal to the viral membrane, are nevertheless highly conserved across strains and among most subtypes. A fundamental question in vaccine design is the extent to which HA stem regions on the surface of the virus are accessible to broadly neutralizing antibodies. Here we report 3D structures derived from cryoelectron tomography of HA on intact 2009 H1N1 pandemic virions in the presence and absence of the antibody C179, which neutralizes viruses expressing a broad range of HA subtypes, including H1, H2, H5, H6, and H9. By fitting previously derived crystallographic structures of trimeric HA into the density maps, we deduced the locations of the molecular surfaces of HA involved in interaction with C179. Using computational methods to distinguish individual unliganded HA trimers from those that have bound C179 antibody, we demonstrate that ∼75% of HA trimers on the surface of the virus have C179 bound to the stem domain. Thus, despite their close packing on the viral membrane, the majority of HA trimers on intact virions are available to bind anti-stem antibodies that target conserved HA epitopes, establishing the feasibility of universal influenza vaccines that elicit such antibodies.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H1N1/química , Modelos Moleculares , Multimerização Proteica , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Pandemias , Estrutura Quaternária de Proteína
6.
IEEE Comput Graph Appl ; 32(5): 39-49, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-24806986

RESUMO

Researchers analyzed and presented volume data from the Visible Human Project (VHP) and data from high-resolution 3D ion-abrasion scanning electron microscopy (IA-SEM). They acquired the VHP data using cryosectioning, a destructive approach to 3D human anatomical imaging resulting in whole-body images with a field of view approaching 2 meters and a minimum resolvable feature size of 300 microns. IA-SEM is a type of block-face imaging microscopy, a destructive approach to microscopic 3D imaging of cells. The field of view of IA-SEM data is on the order of 10 microns (whole cell) with a minimum resolvable feature size of 15 nanometers (single-slice thickness). Despite the difference in subject and scale, the analysis and modeling methods were remarkably similar. They are derived from image processing, computer vision, and computer graphics techniques. Moreover, together we are employing medical illustration, visualization, and rapid prototyping to inform and inspire biomedical science. By combining graphics and biology, we are imaging across nine orders of magnitude of space to better promote public health through research.


Assuntos
Estruturas Celulares/ultraestrutura , Gráficos por Computador , Técnicas Citológicas/métodos , Diagnóstico por Imagem/métodos , Projetos Ser Humano Visível , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imageamento Tridimensional/métodos , Masculino , Camundongos
7.
J Vis Exp ; (58)2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22158337

RESUMO

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) (2). Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor (5,9). The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine. The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Šresolution (9,14). This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Glicoproteínas/química , HIV-1/química , Proteínas do Envelope Viral/química , Microscopia Crioeletrônica/instrumentação , Tomografia com Microscopia Eletrônica/instrumentação , HIV-1/ultraestrutura , Humanos , Modelos Moleculares
8.
J Bacteriol ; 193(6): 1341-50, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21148724

RESUMO

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.


Assuntos
Bdellovibrio/ultraestrutura , Cromossomos Bacterianos/ultraestrutura , Microscopia Crioeletrônica , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/ultraestrutura , Coloração e Rotulagem/métodos
9.
Proc Natl Acad Sci U S A ; 107(30): 13336-41, 2010 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-20624966

RESUMO

The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.


Assuntos
Células Dendríticas/virologia , HIV/fisiologia , Linfócitos T/virologia , Vírion/fisiologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/ultraestrutura , Células Apresentadoras de Antígenos/virologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD4-Positivos/virologia , Comunicação Celular , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Interações Hospedeiro-Patógeno , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura
10.
PLoS Pathog ; 5(9): e1000591, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19779568

RESUMO

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of approximately 150-200 nm, and lengths of up to approximately 5 microm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Macrófagos/ultraestrutura , Macrófagos/virologia , Microscopia Eletrônica de Varredura/métodos , Infecções por HIV/patologia , Humanos , Imageamento Tridimensional , Células Jurkat , Gravação em Vídeo , Vírion/fisiologia
11.
J Struct Biol ; 166(1): 1-7, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19116171

RESUMO

Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and melanocytes at resolutions as high as approximately 6 and approximately 20 nm in the directions parallel and perpendicular, respectively, to the direction of ion-beam milling. The 3D images demonstrate the striking spatial relationships between specific organelles such as mitochondria and membranes of the endoplasmic reticulum, and the distribution of unique cellular components such as melanosomes. We also show that 10nm-sized gold particles and quantum dot particles with 7 nm-sized cores can be detected in single cross-sectional images. IA-SEM is thus a useful tool for imaging large mammalian cells in their entirety at resolutions in the nanometer range.


Assuntos
Células Eucarióticas/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura/métodos , Linhagem Celular Tumoral , Técnicas Citológicas/métodos , Ouro , Humanos , Membranas Intracelulares/ultraestrutura , Melanócitos/ultraestrutura , Melanoma/ultraestrutura , Melanossomas/ultraestrutura , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Organelas/ultraestrutura , Pontos Quânticos , Coloração e Rotulagem
12.
J Biol Chem ; 282(38): 27754-9, 2007 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-17599917

RESUMO

Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design. We used cryoelectron tomography and atomic force microscopy to characterize the structure of an extremely potent HIV-neutralizing protein, D1D2-Ig alpha tp (abbreviated as D1D2-IgP), a polyvalent antibody construct that presents dodecameric CD4 in place of the Fab regions. We show that D1D2-IgP has a novel structure, displaying greater flexibility of its antibody arms than the closely related IgM. Using simian immunodeficiency virus in complex with D1D2-IgP, we present unequivocal evidence that D1D2-IgP can cross-link surface spikes on the same virus and on neighboring viruses. The observed binding to the viral envelope spikes is the result of specific CD4-gp120 interaction, because binding was not observed with MICA-IgP, a construct that is identical to D1D2-IgP except that major histocompatibility complex Class I-related Chain A (MICA) replaces the CD4 moiety. CD4-mediated binding was also associated with a significantly elevated proportion of ruptured viruses. The ratio of inactivated to CD4-liganded gp120-gp41 spikes can be much greater than 1:1, because all gp120-gp41 spikes on the closely apposed surfaces of cross-linked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike cross-linking. These results implicate flexibility rather than steric bulk or polyvalence per se as a structural explanation for the extreme potency of D1D2-IgP and thus suggest polyvalence presented on a flexible scaffold as a key design criterion for small molecule HIV entry inhibitors.


Assuntos
Microscopia Crioeletrônica/métodos , Proteína gp120 do Envelope de HIV/química , Proteínas Virais/química , Animais , Fármacos Anti-HIV/química , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/metabolismo , Humanos , Imageamento Tridimensional , Imunoglobulinas/química , Microscopia de Força Atômica , Modelos Biológicos , Conformação Molecular , Vírus da Imunodeficiência Símia/metabolismo , Tomografia/métodos
14.
J Bacteriol ; 186(15): 5052-61, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15262942

RESUMO

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/ultraestrutura , Escherichia coli/ultraestrutura , Imageamento Tridimensional/métodos , Proteínas de Membrana/metabolismo , Tomografia/métodos , Regulação para Cima , Proteínas de Bactérias/genética , Membrana Celular/fisiologia , Quimiotaxia , Escherichia coli/genética , Escherichia coli/fisiologia , Engenharia Genética/métodos , Proteínas de Membrana/genética , Microscopia Eletrônica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
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