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Optical projection tomography as a tool for 3D microscopy and gene expression studies

SCIENCE 2002年第5567期296卷 541-545页

作者： Sharpe, J Ahlgren, U Perry, P Hill, B Ross, A Hecksher-Sorensen, J Baldock, R Davidson, D Western Gen Hosp MRC Human Genet Unit Edinburgh EH4 2XU Midlothian Scotland

Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal. microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.

关键词： Anatomy, Cross-Sectional Animals DNA-Binding Proteins/analysis Embryo, Mammalian/anatomy & histology Embryo, Mammalian/metabolism Embryonic and Fetal Development Fluorescent Antibody Technique Fluorescent Antibody Technique Gene Expression Gene Expression Profiling/instrumentation Gene Expression Profiling/methods Hepatocyte Nuclear Factor 3-beta image processing, computer-assisted Immunohistochemistry Immunohistochemistry In Situ Hybridization In Situ Hybridization Magnetic Resonance Imaging Mice Microscopy/instrumentation Microscopy/methods Microscopy, Fluorescence/instrumentation Microscopy, Fluorescence/methods Mutation Nervous System/embryology Neurofilament Proteins/analysis Nuclear Proteins/analysis Software Stomach/embryology Tomography Transcription Factors

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Nuclear pore complex structure and dynamics revealed by cryoelectron tomography

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SCIENCE 2004年第5700期306卷 1387-1390页

作者： Beck, M Förster, F Ecke, M Plitzko, JM Melchior, F Gerisch, G Baumeister, W Medalia, O Max Planck Inst Biochem D-82152 Martinsried Germany

Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. To analyze their structure in a close-to-life state, we studied transport-active, intact nuclei from Dictyostelium discoideum by means of cryoelectron tomography. Subvolumes of the tomograms containing individual NPCs were extracted in silico and subjected to three-dimensional classification and averaging, whereby distinct structural states were observed. The central plug/transporter (CP/T) was variable in volume and could occupy different positions, along the nucleocytoplasmic axis, which supports the notion that it essentially represents cargo in transit. Changes in the position of the CP/T were accompanied by structural rearrangements in the NPC scaffold.

关键词： Active Transport, Cell Nucleus Animals Cell Nucleus/ultrastructure Cryoelectron Microscopy Cytoskeleton/ultrastructure Dictyostelium/chemistry Dictyostelium/metabolism Dictyostelium/ultrastructure image processing, computer-assisted Imaging, Three-Dimensional Nuclear Pore/chemistry Nuclear Pore/ultrastructure Tomography/methods

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MICRODOMAINS OF HIGH CALCIUM-CONCENTRATION IN A PRESYNAPTIC TERMINAL

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SCIENCE 1992年第5057期256卷 677-679页

作者： LLINAS, R SUGIMORI, M SILVER, RB MARINE BIOL LAB WOODS HOLEMA 02543 CORNELL UNIV DEPT PHYSIOLITHACANY 14853 CORNELL UNIV PHYSIOL SECTITHACANY 14853

Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion, supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation of a low-affinity calcium-binding site at the active zone.

关键词： Aequorin Animals Calcium/analysis Calcium/metabolism Calcium Channels/metabolism Decapodiformes Electric Stimulation Fluorescent Dyes image processing, computer-assisted Luminescence Luminescence Microscopy Neurotransmitter Agents/secretion Photography Synapses/chemistry Synapses/physiology Synaptic Membranes/metabolism

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T cell receptor signaling precedes immunological synapse formation

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SCIENCE 2002年第5559期295卷 1539-1542页

作者： Lee, KH Holdorf, AD Dustin, ML Chan, AC Allen, PM Shaw, AS Washington Univ Sch Med Dept Pathol & Immunol St Louis MO 63110 USA Skirbal Inst Biomol Med Program Mol Pathogenesis New York NY 10016 USA Genentech Inc Dept Immunol San Francisco CA 94080 USA

The area of contact between a T cell and an antigen-presenting cell (APC) is known as the immunological synapse. Although its exact function is unknown, one model suggests that it allows for T cell receptor (TCR) clustering and for sustained signaling in T cells for many hours. Here we demonstrate that TCR-mediated tyrosine kinase signaling in naive T cells occurred primarily at the periphery of the synapse and was largely abated before mature immunological synapses had formed. These data suggest that many hours of TCR signaling are not required for T cell activation. These observations challenge current ideas about the role of immunological synapses in T cell activation.

关键词： Animals Antigen-Presenting Cells/immunology Cell Division Cells, Cultured Down-Regulation Endocytosis Enzyme Activation image processing, computer-assisted Intercellular Junctions/immunology Intercellular Junctions/metabolism Lymphocyte Activation Lymphocyte Activation Lymphocyte Function-Associated Antigen-1/metabolism Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism Mice Mice, Transgenic Mice, Transgenic Peptides/immunology Protein-Tyrosine Kinases/metabolism Receptor Aggregation Receptors, Antigen, T-Cell/immunology Receptors, Antigen, T-Cell/metabolism Signal Transduction Time Factors T-Lymphocytes/immunology T-Lymphocytes/metabolism ZAP-70 Protein-Tyrosine Kinase

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Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography

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SCIENCE 2002年第5596期298卷 1209-1213页

作者： Medalia, O Weber, I Frangakis, AS Nicastro, D Gerisch, G Baumeister, W Max Planck Inst Biochem D-82152 Martinsried Germany

Electron tomography of vitrifed cells is a noninvasive three-dimensional imaging technique that opens up new vistas for exploring the supramolecular organization of the cytoplasm. We applied this technique to Dictyostelium cells, focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the cross-linking of individual micro laments, their branching angles, and membrane attachment sites can be analyzed. At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26S proteasome, can be identified in an unperturbed cellular environment.

关键词： Actin Cytoskeleton/chemistry Actin Cytoskeleton/metabolism Actin Cytoskeleton/ultrastructure Actins/ultrastructure Animals Binding Sites Cell Membrane/metabolism Cell Membrane/ultrastructure Cell Movement Dictyostelium/chemistry Dictyostelium/physiology Dictyostelium/ultrastructure Endoplasmic Reticulum, Rough/ultrastructure Freezing image processing, computer-assisted Macromolecular Substances Microfilament Proteins/ultrastructure Microfilaments/chemistry Microfilaments/metabolism Microfilaments/ultrastructure Organelles/ultrastructure Peptide Hydrolases/ultrastructure Proteasome Endopeptidase Complex Proteome Protozoan Proteins/ultrastructure Ribosomes/ultrastructure Tomography/methods

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Imaging the distribution and secondary structure of immobilized enzymes using infrared microspectroscopy

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BIOMACROMOLECULES 2003年第1期4卷 70-74页

作者： Mei, Y Miller, L Gao, W Gross, RA Polytech Univ NSF I UCRC Biocatalysis & Bioproc Macromol MetroTech Ctr 6 Brooklyn NY 11201 USA Brookhaven Natl Lab Upton NY 11973 USA

Synchrotron infrared microspectroscopy (SIRMS) was used for the first time to image the distribution and secondary structure of an enzyme (lipase B from Candida antarctica, CALB) immobilized within a macroporous polymer matrix (poly(methyl methacrylate)) at 10 mum resolution. The beads of this catalyst (Novozyme435) were cut into thin sections (12 mum). SIRMS imaging of these thin sections revealed that the enzyme is localized in an external shell of the bead with a thickness of 80-100 mum. Also, the enzyme was unevenly distributed throughout this shell. Furthermore, by SIRMS-generated spectra, it was found that CALB secondary structure was not altered by immobilization. Unlike CALB, polystyrene molecules of similar molecular weight diffuse easily throughout Novozyme435 beads. Scanning electron micrograph (SEM) images of the Novozyme435 beads showed that the average pore size is 10 times larger than CALB or polystyrene molecules, implying that there is no physical barrier to enzyme or substrate diffusion throughout the bead. Thus, the difference between polystyrene and enzyme diffusivity suggests that protein-matrix and protein-protein interactions govern the distribution of the enzyme within the macroporous resin.

关键词： Candida/enzymology Enzymes, Immobilized/chemistry Enzymes, Immobilized/ultrastructure image processing, computer-assisted Lipase/chemistry Lipase/ultrastructure Microscopy, Electron, Scanning Polymethyl Methacrylate Protein Structure, Secondary Sensitivity and Specificity Spectrophotometry, Infrared

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Re-probing of immunoblots after storage for more than a decade

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BIOTECHNIQUES 1997年第2期23卷 250-&页

作者： Gorr, SU Department of Biological and Biophysical Sciences University of Louisville Health Sciences Center KY 40292 USA.

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Assessment of three-dimensional biofilm structure using an optical microscope

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BIOTECHNIQUES 2007年第5期42卷 616-620页

作者： de Carvalho, Carla C. C. R. da Fonseca, M. Manuela R. Univ Tecn Lisboa Ctr Biol & Chem Engn Inst Biotechnol & Bioengn Inst Super Tecn Lisbon 1049001 Portugal

A method allowing the evaluation of the structure and the calculation of the volume of a biofilm, using an optical microscope, is proposed based on the linear relation between the intensity of a pixel in biofilm images grabbed on the x-y plane and the corresponding number of cells in the z direction, which allows the calculation of the biofilm thickness. The method is intended to overcome the need for expensive microscopes to study biofilms.

关键词： Biofilms image processing, computer-assisted Microscopy/instrumentation Optics and Photonics Rhodococcus/cytology Rhodococcus/physiology Solvents

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Virus maturation involving large subunit rotations and local refolding

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SCIENCE 2001年第5517期292卷 744-748页

作者： Conway, JF Wikoff, WR Cheng, N Duda, RL Hendrix, RW Johnson, JE Steven, AC NIAMSD Struct Biol Res Lab Bethesda MD 20892 USA Inst Biol Struct JP Ebel F-38027 Grenoble France Scripps Res Inst Dept Mol Biol La Jolla CA 92037 USA Univ Pittsburgh Dept Biol Sci Pittsburgh PA 15260 USA

Large-scale conformational changes transform viral precursors into infectious virions. The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology. We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II. Rigid-body rotations (similar to 40 degrees) cause switching to an entirely different set of interactions;in addition, two motifs undergo refolding. These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially similar to 40 angstroms apart, close together. The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.

关键词： Amino Acid Motifs Capsid/chemistry Capsid/metabolism Cryoelectron Microscopy Crystallography, X-Ray DNA, Viral/metabolism image processing, computer-assisted Models, Molecular Protein Conformation Protein Folding Protein Precursors/chemistry Protein Precursors/metabolism Protein Structure, Tertiary Protein Subunits Siphoviridae/chemistry Siphoviridae/physiology Siphoviridae/ultrastructure Surface Properties Virus Assembly

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Integrins involved in the adhesion of megakaryocytes to fibronectin and fibrinogen

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BLOOD 1998年第8期92卷 2650-2656页

作者： Schick, PK Wojenski, CM He, X Walker, J Marcinkiewicz, C Niewiarowski, S Thomas Jefferson Univ Jefferson Med Coll Cardeza Fdn Hematol Res Philadelphia PA 19107 USA Thomas Jefferson Univ Jefferson Med Coll Dept Med Philadelphia PA 19107 USA Thomas Jefferson Univ Jefferson Med Coll Dept Mol Pharmacol & Biochem Philadelphia PA 19107 USA Temple Univ Sch Med Dept Physiol Philadelphia PA 19122 USA

We studied integrins involved in the adhesion of resting and activated megakaryocytes (MK) to fibronectin (FN) and fibrinogen (FGN). Guinea pig MK were isolated and in some experiments were activated by thrombin, MK adhering to FN or FGN coated on coverslips were quantitated by a computerized image analysis program. The binding of soluble human FN to MK was detected by Western blotting, Antiintegrin antibodies, disintegrins, and cyclic RGD peptides were used to identify integrins involved in the adhesion of MK to FN or FGN, Resting MK adhered to coverslips with immobilized FN. The adhesion of MK to FN was primarily inhibited by an anti-alpha 5 antibody and EMF-10, a distintegrin highly specific for alpha 5 beta 1. However, the adhesion of MK to FN was not blocked by agents that inhibit alpha IIb beta 3, alpha v beta 3 or alpha 4 beta 1. A pr activating antibody increased the number of MK bound to FN due to the activation of alpha 5 beta 1. The binding of soluble FN was also primarily inhibited by agents that block alpha 5 beta 1, Resting MK did not adhere to FGN. However, MK activated by thrombin did adhere to FGN, This binding was mediated by alpha IIb beta 3, because binding was inhibited by bitistatin, a disintegrin, and a cyclic RGD peptide that are known to block this integrin. The binding of thrombin-activated MK to FN was mediated by both alpha 5 beta 1 and alpha IIb beta 3 based on the additive effect of agents that inhibit these integrins. The study indicates that resting MK bind to FN but not to FGN and that alpha 5 beta 1 is the major integrin involved in the binding of MK to FN. Activated MK bind to FGN primarily by alpha IIb beta 3. However, the binding of activated MK to FN is due to both alpha 5 beta 1 and alpha IIb beta 3. The demonstration that alpha 5 beta 1 and that alpha IIb beta 3 are involved in MK adhesion indicates that these integrins may have a role in MK maturation and platelet production. (C) 1998 by The American Society of Hematology.

关键词： Animals Cell Adhesion/drug effects Cell Adhesion/physiology Disintegrins/pharmacology Edetic Acid/pharmacology Edetic Acid/pharmacology Fibrinogen/metabolism Fibronectins/metabolism Guinea Pigs Humans image processing, computer-assisted Megakaryocytes/cytology Megakaryocytes/drug effects Peptides/pharmacology Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors Platelet Glycoprotein GPIIb-IIIa Complex/physiology Receptors, Fibronectin/antagonists & inhibitors Receptors, Fibronectin/physiology Thrombin/pharmacology Vascular Cell Adhesion Molecule-1/metabolism Viper Venoms/pharmacology Vitronectin/metabolism

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