Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites
Publication Date
February 24, 2015
Journal
PLOS Pathogens
Authors
Jens Prescher, Viola Baumgärtel, Sergey Ivanchenko, Adriano A. Torrano, et al
Volume
11
Issue
2
Pages
e1004677
DOI
https://dx.plos.org/10.1371/journal.ppat.1004677
Publisher URL
http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1004677
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/25710462
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4339578
Europe PMC
http://europepmc.org/abstract/MED/25710462
Web of Science
000352083400051
Scopus
84924352677
Mendeley
http://www.mendeley.com/research/superresolution-imaging-escrtproteins-hiv1-assembly-sites
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Mendeley | Further Information

{"title"=>"Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites", "type"=>"journal", "authors"=>[{"first_name"=>"Jens", "last_name"=>"Prescher", "scopus_author_id"=>"55865128100"}, {"first_name"=>"Viola", "last_name"=>"Baumgärtel", "scopus_author_id"=>"20336622900"}, {"first_name"=>"Sergey", "last_name"=>"Ivanchenko", "scopus_author_id"=>"6602149599"}, {"first_name"=>"Adriano A.", "last_name"=>"Torrano", "scopus_author_id"=>"55915761900"}, {"first_name"=>"Christoph", "last_name"=>"Bräuchle", "scopus_author_id"=>"7005564729"}, {"first_name"=>"Barbara", "last_name"=>"Müller", "scopus_author_id"=>"7403187010"}, {"first_name"=>"Don C.", "last_name"=>"Lamb", "scopus_author_id"=>"7202727460"}], "year"=>2015, "source"=>"PLoS Pathogens", "identifiers"=>{"pui"=>"602901909", "sgr"=>"84924352677", "issn"=>"15537374", "pmid"=>"25710462", "scopus"=>"2-s2.0-84924352677", "doi"=>"10.1371/journal.ppat.1004677", "isbn"=>"1553-7374"}, "id"=>"129a344c-fa86-3c00-9a14-3d0c297bf1e1", "abstract"=>"The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley's L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.", "link"=>"http://www.mendeley.com/research/superresolution-imaging-escrtproteins-hiv1-assembly-sites", "reader_count"=>67, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Student > Doctoral Student"=>3, "Researcher"=>12, "Student > Ph. D. Student"=>28, "Student > Master"=>9, "Other"=>2, "Student > Bachelor"=>5, "Professor"=>4}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Student > Doctoral Student"=>3, "Researcher"=>12, "Student > Ph. D. Student"=>28, "Student > Master"=>9, "Other"=>2, "Student > Bachelor"=>5, "Professor"=>4}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>14, "Agricultural and Biological Sciences"=>36, "Medicine and Dentistry"=>1, "Neuroscience"=>1, "Physics and Astronomy"=>1, "Chemistry"=>5, "Immunology and Microbiology"=>4}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>5}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>4}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>36}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>14}, "Unspecified"=>{"Unspecified"=>4}}, "reader_count_by_country"=>{"United States"=>1, "United Kingdom"=>1, "France"=>2, "Switzerland"=>1}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1922940"], "description"=>"<p>HeLa cells were transfected with HIV:HIV<sup>mCherry</sup> and endogenous CHMP2A was detected by immunostaining 14–15 h post transfection. HeLa cells were imaged using TIRFM. (<b>A</b>) <i>Left panel</i>. A merged time-projected TIRF image of HIV<sup>mCherry</sup> (magenta) and CHMP2A (green) prior to STORM analysis. <i>Middle panel</i>. A zoomed-in image of the selected CHMP2A cluster highlighted in grey in the left panel colocalizing with an HIV assembly site. <i>Right panel</i>. The corresponding drift-corrected STORM image of the CHMP2A cluster. Scale bars: 500 nm. (<b>B</b>) The size distribution of all CHMP2A structures colocalizing with HIV<sup>mCherry</sup> with an average cluster size (FWHM) of 56 ± 12 nm. (<b>C</b>) Size distribution of all non-colocalizing CHMP2A clusters in cells co-expressing HIV<sup>mCherry</sup> with an average cluster size (FWHM) of 59 ± 20 nm.</p>", "links"=>[], "tags"=>["drive HIV release", "2a", "colocalizing ESCRT clusters", "ha", "membrane scission process", "ESCRT structures colocalizing", "assembly", "CHMP 4B", "ESCRT protein assemblies", "bud", "CHMP 2 form", "alix", "colocalizing ESCRT complexes", "fusion protein tag"], "article_id"=>1317036, "categories"=>["Biological Sciences"], "users"=>["Jens Prescher", "Viola Baumgärtel", "Sergey Ivanchenko", "Adriano A. Torrano", "Christoph Bräuchle", "Barbara Müller", "Don C. Lamb"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1004677.g005", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Super_resolution_imaging_of_CHMP2A_at_HIV_1_assembly_sites_/1317036", "title"=>"Super-resolution imaging of CHMP2A at HIV-1 assembly sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:23:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922936"], "description"=>"<p>HeLa cells were transfected with HIV:HIV<sup>mCherry</sup> and endogenous ALIX was immunostained at 14 – 15 h post transfection. Cells were imaged by TIRFM. (<b>A</b>) A time projected image of ALIX (green) overlaid with TIRF image of HIV<sup>mCherry</sup> assembly sites (magenta) is shown. Circles indicate non-colocalizing ALIX clusters and the rectangles highlight different colocalized ALIX classes. The crops show drift-corrected super-resolution STORM images of the three identified object classes: condensed ALIX structures without a surrounding cloud (1), ALIX structures with a central, condensed spot surrounded by a cloud-like structure (2) and diffuse ALIX membrane assemblies without a central spot (3). Scale bars: 10 μm (large image), 100 nm (crops). (<b>B</b>) Size distribution of all central spots of colocalizing ALIX protein clusters with an average cluster size (FWHM) of 64 ± 18 nm. (<b>C</b>) Size distribution of all central spots of non-colocalizing ALIX protein clusters with an average cluster size (FWHM) of 74 ± 26 nm. (<b>D</b>) Cloud cluster size characterization for all cloud structures of ALIX colocalizing with HIV<sup>mCherry</sup> obtained by Ripley’s cluster analysis with an average diameter of 164 ± 31 nm. (<b>E</b>) The distribution of the minimum number of ALIX proteins detected per cloud estimated from the super-resolution data.</p>", "links"=>[], "tags"=>["drive HIV release", "2a", "colocalizing ESCRT clusters", "ha", "membrane scission process", "ESCRT structures colocalizing", "assembly", "CHMP 4B", "ESCRT protein assemblies", "bud", "CHMP 2 form", "alix", "colocalizing ESCRT complexes", "fusion protein tag"], "article_id"=>1317033, "categories"=>["Biological Sciences"], "users"=>["Jens Prescher", "Viola Baumgärtel", "Sergey Ivanchenko", "Adriano A. Torrano", "Christoph Bräuchle", "Barbara Müller", "Don C. Lamb"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1004677.g003", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Super_resolution_imaging_of_endogenous_ALIX_at_HIV_1_assembly_sites_/1317033", "title"=>"Super-resolution imaging of endogenous ALIX at HIV-1 assembly sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:23:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922930"], "description"=>"<p>HeLa cells were transfected with HIV:HIV<sup>mCherry</sup> and endogenous Tsg101 was immunostained at 14 – 15 h post transfection. (<b>A</b>) Cells were imaged using TIRFM. A time projection of the TIRFM images are shown (HIV<sup>mCherry</sup>, <i>left panel</i>; Tsg101, <i>middle panel</i>; Overlay, magenta: HIV<sup>mCherry</sup>, green: Tsg101, <i>right panel</i>), scale bars: 10 μm. (<b>B</b>) <i>Left panel</i>. A zoomed image of the region highlighted in panel <b>A</b>. Scale bar: 2 μm. <i>Middle panel</i>. A zoomed image of the individual colocalizing Tsg101 cluster highlighted in the left panel. Scale bar: 500 nm. <i>Right panel</i>. A drift-corrected STORM image of the Tsg101 complex shown in the middle panel. Scale bar: 500 nm. (<b>C</b>) The size distribution of all Tsg101 structures colocalizing with HIV<sup>mCherry</sup>, revealing an average cluster size of 58 ± 7 nm. (<b>D</b>) Size distribution of all non-colocalizing Tsg101 clusters in cells expressing HIV<sup>mCherry</sup> revealing an average cluster size of 60 ± 15 nm or (<b>E</b>) or in cell expressing HIV<sup>mCherry</sup> late- revealing average cluster size of 58 ± 16 nm. Number <i>N</i> represents the number events contributing to the respective histogram.</p>", "links"=>[], "tags"=>["drive HIV release", "2a", "colocalizing ESCRT clusters", "ha", "membrane scission process", "ESCRT structures colocalizing", "assembly", "CHMP 4B", "ESCRT protein assemblies", "bud", "CHMP 2 form", "alix", "colocalizing ESCRT complexes", "fusion protein tag"], "article_id"=>1317031, "categories"=>["Biological Sciences"], "users"=>["Jens Prescher", "Viola Baumgärtel", "Sergey Ivanchenko", "Adriano A. Torrano", "Christoph Bräuchle", "Barbara Müller", "Don C. Lamb"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1004677.g002", "stats"=>{"downloads"=>0, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Super_resolution_images_of_Tsg101_at_HIV_1_assembly_sites_/1317031", "title"=>"Super-resolution images of Tsg101 at HIV-1 assembly sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:23:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922929"], "description"=>"<p>(<b>A</b>) A simplified schematic of the interaction scheme between HIV-1 assembly sites and the ESCRT recruitment pathway. The constructs analyzed in this study are highlighted in purple. (<b>B</b>) An overview of the proposed models of membrane scission where ESCRT factors interact either via external constriction from the cytosol (cytosolic model, Model 1), via internal constrict of the budding virus in the neck region (neck model, Model 2) or from within the bud (bud model, Model 3).</p>", "links"=>[], "tags"=>["drive HIV release", "2a", "colocalizing ESCRT clusters", "ha", "membrane scission process", "ESCRT structures colocalizing", "assembly", "CHMP 4B", "ESCRT protein assemblies", "bud", "CHMP 2 form", "alix", "colocalizing ESCRT complexes", "fusion protein tag"], "article_id"=>1317030, "categories"=>["Biological Sciences"], "users"=>["Jens Prescher", "Viola Baumgärtel", "Sergey Ivanchenko", "Adriano A. Torrano", "Christoph Bräuchle", "Barbara Müller", "Don C. Lamb"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1004677.g001", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gag_ESCRT_Interactions_/1317030", "title"=>"Gag-ESCRT Interactions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:23:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922955", "https://ndownloader.figshare.com/files/1922956", "https://ndownloader.figshare.com/files/1922957", "https://ndownloader.figshare.com/files/1922958", "https://ndownloader.figshare.com/files/1922959", "https://ndownloader.figshare.com/files/1922960", "https://ndownloader.figshare.com/files/1922961", "https://ndownloader.figshare.com/files/1922962", "https://ndownloader.figshare.com/files/1922963", "https://ndownloader.figshare.com/files/1922964", "https://ndownloader.figshare.com/files/1922965", "https://ndownloader.figshare.com/files/1922966", "https://ndownloader.figshare.com/files/1922967", "https://ndownloader.figshare.com/files/1922968"], "description"=>"<div><p>The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.</p></div>", "links"=>[], "tags"=>["drive HIV release", "2a", "colocalizing ESCRT clusters", "ha", "membrane scission process", "ESCRT structures colocalizing", "assembly", "CHMP 4B", "ESCRT protein assemblies", "bud", "CHMP 2 form", "alix", "colocalizing ESCRT complexes", "fusion protein tag"], "article_id"=>1317049, "categories"=>["Biological Sciences"], "users"=>["Jens Prescher", "Viola Baumgärtel", "Sergey Ivanchenko", "Adriano A. Torrano", "Christoph Bräuchle", "Barbara Müller", "Don C. Lamb"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1004677.s001", "https://dx.doi.org/10.1371/journal.ppat.1004677.s002", "https://dx.doi.org/10.1371/journal.ppat.1004677.s003", "https://dx.doi.org/10.1371/journal.ppat.1004677.s004", "https://dx.doi.org/10.1371/journal.ppat.1004677.s005", "https://dx.doi.org/10.1371/journal.ppat.1004677.s006", "https://dx.doi.org/10.1371/journal.ppat.1004677.s007", "https://dx.doi.org/10.1371/journal.ppat.1004677.s008", "https://dx.doi.org/10.1371/journal.ppat.1004677.s009", "https://dx.doi.org/10.1371/journal.ppat.1004677.s010", "https://dx.doi.org/10.1371/journal.ppat.1004677.s011", "https://dx.doi.org/10.1371/journal.ppat.1004677.s012", "https://dx.doi.org/10.1371/journal.ppat.1004677.s013", "https://dx.doi.org/10.1371/journal.ppat.1004677.s014"], "stats"=>{"downloads"=>22, "page_views"=>40, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Super_Resolution_Imaging_of_ESCRT_Proteins_at_HIV_1_Assembly_Sites_/1317049", "title"=>"Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-02-24 03:23:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922938"], "description"=>"<p>HeLa cells were transfected with both HIV:HIV<sup>mCherry</sup> and CHMP4B-HA and CHMP4B-HA was detected by immunostaining 14–15 h post transfection. Cells were imaged by TIRFM. (<b>A</b>) <i>Left panel</i>. A time projected overlay of HeLa cells transfected with HIV<sup>mCherry</sup> (magenta) and CHMP4B-HA (green). The circles indicate partially diffuse CHMP4B clusters at the membrane, which are ignored in further analyses. Scale bar: 10 μm. <i>Middle panel</i>. A zoomed-in image of a selected CHMP4B-HA cluster (highlighted in grey in the left panel) colocalizing with HIV<sup>mCherry</sup>. <i>Right panel</i>. The corresponding drift-corrected STORM image. Scale bars: 500 nm (<b>B</b>) Size distribution of all CHMP4B-HA structures colocalizing with HIV<sup>mCherry</sup> with an average cluster size (FWHM) of 56 ± 12 nm. (<b>C</b>) Size distribution of all non-colocalizing CHMP4B-HA clusters in cells co-expressing HIV<sup>mCherry</sup> wildtype with an average cluster size (FWHM) of 69 ± 30 nm. (<b>D</b>) Size distribution of all non-colocalizing CHMP4B clusters in cells co-expressing HIV<sup>mCherry</sup> late- with an average cluster size (FWHM) of 53 ± 16 nm.</p>", "links"=>[], "tags"=>["drive HIV release", "2a", "colocalizing ESCRT clusters", "ha", "membrane scission process", "ESCRT structures colocalizing", "assembly", "CHMP 4B", "ESCRT protein assemblies", "bud", "CHMP 2 form", "alix", "colocalizing ESCRT complexes", "fusion protein tag"], "article_id"=>1317034, "categories"=>["Biological Sciences"], "users"=>["Jens Prescher", "Viola Baumgärtel", "Sergey Ivanchenko", "Adriano A. Torrano", "Christoph Bräuchle", "Barbara Müller", "Don C. Lamb"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1004677.g004", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Super_resolution_imaging_of_CHMP4B_HA_clusters_/1317034", "title"=>"Super-resolution imaging of CHMP4B-HA clusters.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:23:39"}

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  • {"unique-ip"=>"14", "full-text"=>"15", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"6", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"20", "full-text"=>"19", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
  • {"unique-ip"=>"12", "full-text"=>"11", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}

Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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