3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography
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{"title"=>"3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography", "type"=>"journal", "authors"=>[{"first_name"=>"Mike", "last_name"=>"Reichelt", "scopus_author_id"=>"56992691200"}, {"first_name"=>"Lydia", "last_name"=>"Joubert", "scopus_author_id"=>"8245672400"}, {"first_name"=>"John", "last_name"=>"Perrino", "scopus_author_id"=>"15026929300"}, {"first_name"=>"Ai Leen", "last_name"=>"Koh", "scopus_author_id"=>"24174519000"}, {"first_name"=>"Ibanri", "last_name"=>"Phanwar", "scopus_author_id"=>"35726913400"}, {"first_name"=>"Ann M.", "last_name"=>"Arvin", "scopus_author_id"=>"26425414800"}], "year"=>2012, "source"=>"PLoS Pathogens", "identifiers"=>{"sgr"=>"84864044838", "doi"=>"10.1371/journal.ppat.1002740", "pui"=>"365284289", "issn"=>"15537366", "pmid"=>"22685402", "isbn"=>"1553-7374", "scopus"=>"2-s2.0-84864044838"}, "id"=>"2cb519cb-545b-3bb7-a425-1cd4e98fc1ee", "abstract"=>"Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.", "link"=>"http://www.mendeley.com/research/3d-reconstruction-vzv-infected-cell-nuclei-pml-nuclear-cages-serial-section-array-scanning-electron", "reader_count"=>44, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>2, "Researcher"=>15, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>2, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>4, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>2, "Researcher"=>15, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>2, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>4, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>29, "Medicine and Dentistry"=>1, "Neuroscience"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>1, "Earth and Planetary Sciences"=>1, "Linguistics"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Neuroscience"=>{"Neuroscience"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>29}, "Computer Science"=>{"Computer Science"=>1}, "Linguistics"=>{"Linguistics"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>4}}, "reader_count_by_country"=>{"Netherlands"=>1, "United States"=>2, "Japan"=>1, "Taiwan"=>1, "France"=>1}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/326970", "https://ndownloader.figshare.com/files/327035", "https://ndownloader.figshare.com/files/327092", "https://ndownloader.figshare.com/files/327165", "https://ndownloader.figshare.com/files/327228", "https://ndownloader.figshare.com/files/327287", "https://ndownloader.figshare.com/files/327336", "https://ndownloader.figshare.com/files/327393", "https://ndownloader.figshare.com/files/327451", "https://ndownloader.figshare.com/files/327504", "https://ndownloader.figshare.com/files/327582", "https://ndownloader.figshare.com/files/327641", "https://ndownloader.figshare.com/files/327711", "https://ndownloader.figshare.com/files/327772"], "description"=>"<div><p>Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) <em>in vitro</em> and in human dorsal root ganglia and skin xenografts <em>in vivo</em>. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.</p> </div>", "links"=>[], "tags"=>["3d", "reconstruction", "vzv", "infected", "nuclei", "pml", "cages", "serial", "scanning", "electron", "microscopy", "tomography"], "article_id"=>124519, "categories"=>["Cancer", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.s001", "https://dx.doi.org/10.1371/journal.ppat.1002740.s002", "https://dx.doi.org/10.1371/journal.ppat.1002740.s003", "https://dx.doi.org/10.1371/journal.ppat.1002740.s004", "https://dx.doi.org/10.1371/journal.ppat.1002740.s005", "https://dx.doi.org/10.1371/journal.ppat.1002740.s006", "https://dx.doi.org/10.1371/journal.ppat.1002740.s007", "https://dx.doi.org/10.1371/journal.ppat.1002740.s008", "https://dx.doi.org/10.1371/journal.ppat.1002740.s009", "https://dx.doi.org/10.1371/journal.ppat.1002740.s010", "https://dx.doi.org/10.1371/journal.ppat.1002740.s011", "https://dx.doi.org/10.1371/journal.ppat.1002740.s012", "https://dx.doi.org/10.1371/journal.ppat.1002740.s013", "https://dx.doi.org/10.1371/journal.ppat.1002740.s014"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/3D_Reconstruction_of_VZV_Infected_Cell_Nuclei_and_PML_Nuclear_Cages_by_Serial_Section_Array_Scanning_Electron_Microscopy_and_Electron_Tomography/124519", "title"=>"3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-06-07 01:15:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/627124"], "description"=>"<p>(A–F) Melanoma cells that expressed doxycycline-induced PML IV were infected with VZV for 48 hours. (A) Analysis by confocal microscopy: permeabilized cells on coverslips were immunostained for PML (green) and ORF23 capsid protein (red); nuclei were stained with Hoechst (blue). Scale bar, 10 µm. (B) Immunogold TEM analysis: cells were high-pressure frozen, freeze-substituted, embedded in LR-White resin and then labeled with anti-PML polyclonal rabbit antibody and Protein A conjugated with 15 nm gold particles. Note the dense PML-gold labeling (arrows) in the amorphous layer (surrounded by a green line) that encloses the sequestered capsids. (C and D). Standard TEM for morphological analysis: cells were aldehyde-fixed, ‘en block’ stained for high contrast and then embedded in epoxy-resin. Note the electron dense amorphous PML layer (surrounded by green line) that encloses the clustered capsids. (D) Three types of capsids (A, B, C-type capsids, red arrows) can be distinguished by TEM. (E and F) Scanning EM analysis with a back-scattered electron detector (BSE) of the same sample as in C. Note the electron dense PML layer (surrounded by a green line) that encloses the sequestered capsids. (F) The three types of capsids (A, B, C-type capsids, red arrows) can also be distinguished by BSE-SEM. Scale bars in B–F are 500 nm.</p>", "links"=>[], "tags"=>["pml", "cages", "vzv", "nucleocapsids", "confocal", "tem"], "article_id"=>297611, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Visualization_of_PML_cages_and_VZV_nucleocapsids_by_confocal_microscopy_TEM_and_SEM_/297611", "title"=>"Visualization of PML cages and VZV nucleocapsids by confocal microscopy, TEM and SEM.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-07 02:06:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/627296"], "description"=>"<p>SSA-SEM enables the three-dimensional reconstruction of cell nuclei and PML domains combined with the visualization and quantification of VZV capsids with ultrastructural precision. (A) Ribbons of ultrathin serial sections are placed on gelatin-coated glass slides and then carbon-coated to prevent charging effects during SEM imaging. The indicated area (red square) contains about 60 consecutive sections. A standard TEM slot-grid (arrow) commonly used in serial section TEM and a ten-cent coin are shown for size comparison. (B) Low magnification view of a ribbon of serial sections imaged by SEM using a back-scattered electron detector (BSE). (C) Using BSE-SEM, regions of interest (ROI), such as whole cells, nuclei or PML-domains can be identified and then repeatedly imaged in consecutive sections, yielding a stack of unaligned digital images of the ROI. (D) The stack of digital images must be aligned, either manually or automatically, for later 3D reconstruction. (E) Structures of interest, such as electron dense heterochromatin (blue), PML domains (green) and VZV capsids (yellow) are manually or automatically (threshold) traced in each serial section for quantification of numbers, areas or volumes and for the visualization of size, shape and distribution of segmented structures in the final 3D model (F).</p>", "links"=>[], "tags"=>["serial", "scanning", "electron", "microscopy"], "article_id"=>297788, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Outline_of_the_serial_section_array_scanning_electron_microscopy_SSA_SEM_method_/297788", "title"=>"Outline of the serial section array scanning electron microscopy (SSA-SEM) method.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-07 02:09:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/627465"], "description"=>"<p>Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s001\" target=\"_blank\">Video S1</a>. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s003\" target=\"_blank\">Video S3</a>. All scale bars are 5 µm.</p>", "links"=>[], "tags"=>["vzv", "nucleocapsids", "nuclei", "pml"], "article_id"=>297953, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Three_dimensional_distribution_of_VZV_nucleocapsids_in_cell_nuclei_without_PML_cages_/297953", "title"=>"Three-dimensional distribution of VZV nucleocapsids in cell nuclei without PML cages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-07 02:12:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/627639"], "description"=>"<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) BSE-SEM images at different magnifications of a syncytium of VZV infected melanoma cells. Left panel: low magnification view of a syncytium; middle panel: one nucleus of the same syncytium with two PML cages; right panel: higher magnification view of a PML cage with sequestered VZV capsids. Black squares indicate areas that are shown at higher magnification in the panels to the right. Scale bars are 5 µm. (B) Five representative images (s1, s5, s9, s13, s17) from a series of 18 consecutive sections through the nucleus shown in A, middle panel. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s004\" target=\"_blank\">Video S4</a>. (C and D) 3D models based on tracing and segmentation in all 18 sections of electron dense heterochromatin (blue); nucleocapsids (yellow spheres) and PML cages (green, shown only in D). 1,732 and 1,324 capsids were identified in the upper and lower PML cage, respectively). Scale bars are 5 µm. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s005\" target=\"_blank\">Video S5</a>. (E and F) 3D models of the upper PML cage. Color code as above, but immature capsids (A and B-type capsids) are shown as yellow spheres and mature capsids (C-type) in orange; the PML cage is transparent green (shown only in F). Scale bars are 2 µm.</p>", "links"=>[], "tags"=>["vzv", "nucleocapsids", "nuclei", "pml"], "article_id"=>298128, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Three_dimensional_distribution_of_VZV_nucleocapsids_in_host_cell_nuclei_with_PML_cages_/298128", "title"=>"Three-dimensional distribution of VZV nucleocapsids in host cell nuclei with PML cages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-07 02:15:28"}
  • {"files"=>["https://ndownloader.figshare.com/files/627817"], "description"=>"<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s006\" target=\"_blank\">Video S6</a>. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s007\" target=\"_blank\">Video S7</a>. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s008\" target=\"_blank\">Video S8</a>.</p>", "links"=>[], "tags"=>["volume-reconstruction", "vzv", "infected", "nucleus", "pml"], "article_id"=>298304, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Large_volume_reconstruction_of_a_VZV_infected_cell_nucleus_with_four_PML_cages_/298304", "title"=>"Large volume-reconstruction of a VZV infected cell nucleus with four PML cages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-07 02:18:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/627980"], "description"=>"<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 hours and then high pressure frozen, freeze-substituted, embedded in LR-White resin and labeled with anti-PML polyconal rabbit antibody and Protein A conjugated with 15 nm gold particles. (A) A representative TEM image from a series of seven consecutive 100 nm sections is shown. The area in the blue square (left panel) is shown at higher magnification in the right panel. PML specific gold labeling (green arrows) identifies the PML cage (surrounded by a green line, left panel) in the nucleus. Scale bars are 500 nm. (B) The 3D model shows the electron dense heterochromatin (blue) and the location of mature capsids (63; red spheres), immature capsids (403; yellow spheres) and all PML-specific gold particles (5,219; small green spheres) that were identified in the serial sections. Entrapped mature capsids with associated PML labeling are shown as red/green spheres and immature capsids with PML labeling are shown as yellow/green spheres. (C) Same 3D model as in B but at higher magnification and in a different angle. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s009\" target=\"_blank\">Video S9</a>.</p>", "links"=>[], "tags"=>["entrapped", "vzv", "capsids", "pml"], "article_id"=>298463, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PML_protein_is_associated_with_entrapped_VZV_capsids_inside_PML_cages_/298463", "title"=>"PML protein is associated with entrapped VZV capsids inside PML cages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-07 02:21:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/628147"], "description"=>"<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s010\" target=\"_blank\">Video S10</a>. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s011\" target=\"_blank\">Video S11</a>. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).</p>", "links"=>[], "tags"=>["tomography", "pml", "cages", "reveals", "cross-linking", "vzv", "capsids", "electron-dense"], "article_id"=>298625, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Electron_tomography_of_PML_cages_reveals_the_cross_linking_of_VZV_capsids_by_an_electron_dense_meshwork_/298625", "title"=>"Electron tomography of PML cages reveals the cross-linking of VZV capsids by an electron-dense meshwork.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-07 02:23:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/628268"], "description"=>"<p>The number of free or sequestered nucleocapsids identified in VZV infected host cell nuclei and the volume of the analyzed nuclei and PML cages were determined by counting the corresponding traces in all serial sections that were used to generate the 3D models. Capsid densities were calculated by dividing the number of nucleocapsids with the corresponding volume (µm<sup>3</sup>) of the PML cage or nucleus. The left column indicates the corresponding figures where the quantified objects are visualized.</p>", "links"=>[], "tags"=>["structures", "3d", "reconstructions", "vzv", "infected"], "article_id"=>298756, "categories"=>["Virology", "Microbiology"], "users"=>["Mike Reichelt", "Lydia Joubert", "John Perrino", "Ai Leen Koh", "Ibanri Phanwar", "Ann M. Arvin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002740.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_structures_identified_in_3D_reconstructions_of_VZV_infected_host_cell_nuclei_/298756", "title"=>"Quantification of structures identified in 3D reconstructions of VZV infected host cell nuclei.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-06-07 02:25:56"}

PMC Usage Stats | Further Information

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