A New Role for Clathrin Adaptor Proteins 1 and 3 in Lipoplex Trafficking
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{"title"=>"A new role for clathrin adaptor proteins 1 and 3 in lipoplex trafficking", "type"=>"journal", "authors"=>[{"first_name"=>"Justine E.", "last_name"=>"Alford", "scopus_author_id"=>"56097566600"}, {"first_name"=>"Jade", "last_name"=>"Gumbs", "scopus_author_id"=>"56096657900"}, {"first_name"=>"Emma C.", "last_name"=>"Anderson", "scopus_author_id"=>"26655263200"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"372764683", "sgr"=>"84897506645", "pmid"=>"24618578", "scopus"=>"2-s2.0-84897506645", "doi"=>"10.1371/journal.pone.0091429", "issn"=>"19326203"}, "id"=>"6c780922-f746-3945-ba37-382ff9d1391b", "abstract"=>"Intracellular protein trafficking through secretory and endocytic pathways depends on the function of adaptor proteins that bind motifs on cargo proteins. The adaptor proteins then recruit coat proteins such as clathrin, enabling the formation of a transport vesicle. While studying the role of the clathrin adaptor proteins, AP-1, AP-2 and AP-3 in viral protein trafficking, we discovered that AP-1 and AP-3 potentially have a role in successful transfection of mammalian cells with DNA-liposome complexes (lipoplexes). We showed that AP-1, -2 and -3 are not required for lipoplexes to enter cells, but that lipoplexes and/or released DNA are unable to reach the nucleus in the absence of AP-1 or AP-3, leading to minimal exogenous gene expression. In contrast, gene expression from liposome-delivered mRNA, which does not require nuclear entry, was not impaired by the absence of AP-1 or AP-3. Despite the use of lipoplexes to mediate gene delivery being so widely used in cell biology and, more recently, gene therapy, the mechanism by which lipoplexes or DNA reach the nucleus is poorly characterised. This work sheds light on the components involved in this process, and demonstrates a novel role for AP-1 and AP-3 in trafficking lipoplexes.", "link"=>"http://www.mendeley.com/research/new-role-clathrin-adaptor-proteins-1-3-lipoplex-trafficking", "reader_count"=>9, "reader_count_by_academic_status"=>{"Researcher"=>2, "Student > Ph. D. Student"=>2, "Student > Master"=>1, "Student > Bachelor"=>3, "Professor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>2, "Student > Ph. D. Student"=>2, "Student > Master"=>1, "Student > Bachelor"=>3, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>5, "Medicine and Dentistry"=>1, "Neuroscience"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Neuroscience"=>{"Neuroscience"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}}, "reader_count_by_country"=>{"Japan"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1415337"], "description"=>"<p><b>A.</b> Relative luciferase activity of lysates from cells transfected with a <i>Renilla</i> luciferase plasmid 24 hours after siRNAs (grey bars) or co-transfected with plasmid and siRNAs (black bars). The data are from three or four independent experiments, error bars represent the standard error of the mean and asterisks indicate significant differences from no siRNA (−) samples (p = 9.5×10<sup>−5</sup> and 5.1×10<sup>−4</sup> for prior transfection of AP-1 and AP-3 siRNAs respectively). <b>B.</b> GFP fluorescence (green) of cells transfected with a GFP plasmid 24 hours after siRNAs. DAPI nuclear stain is shown in blue and 50 µm scale bars are shown in each image. The percentage of GFP positive cells was counted in ten fields of view (1000–2000 cells) for each siRNA treatment, from up to four independent experiments. The mean percentage of GFP positive cells relative to the no siRNA samples (−), +/− standard error, is shown. Asterisks indicate significant differences from no siRNA (−) samples (p = 2.7×10<sup>−11</sup> and 5.4×10<sup>−11</sup> for AP-1 and AP-3 siRNAs respectively).</p>", "links"=>[], "tags"=>["biotechnology", "Genetic engineering", "Computational biology", "Molecular genetics", "gene expression", "genetics", "microbiology", "Virology", "Viral transmission and infection", "Viral entry", "Molecular cell biology", "Cellular structures", "cytoplasm", "Membranes and sorting", "Nucleic acids", "knockdown", "ap-1", "ap-3", "exogenous"], "article_id"=>958165, "categories"=>["Biological Sciences"], "users"=>["Justine E. Alford", "Jade Gumbs", "Emma C. Anderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0091429.g002", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_siRNA_mediated_knockdown_of_AP_1_or_AP_3_reduces_exogenous_gene_expression_/958165", "title"=>"siRNA-mediated knockdown of AP-1 or AP-3 reduces exogenous gene expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-03-11 03:15:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1415341"], "description"=>"<p>HeLa cells were transfected with no siRNA (−, <b>A</b>), siRNAs against AP-1γ, AP-2 µ or AP-3δ (<b>B–D</b> respectively) or a control siRNA (con,<b>E</b>). 24 hours later, the cells were transfected with GFP plasmid using a rhodamine B-labelled lipofection reagent, and fixed after 6 hours. Rhodamine fluorescence from METAFECTENE®-FluoR (red) is shown in the left panels, GFP fluorescence (green) is shown in the middle panels, and a merged image of the two channels is shown in the right panels. 25 µm scale bars are highlighted in the bottom right of the merged images.</p>", "links"=>[], "tags"=>["biotechnology", "Genetic engineering", "Computational biology", "Molecular genetics", "gene expression", "genetics", "microbiology", "Virology", "Viral transmission and infection", "Viral entry", "Molecular cell biology", "Cellular structures", "cytoplasm", "Membranes and sorting", "Nucleic acids", "ap-3", "cellular", "uptake"], "article_id"=>958169, "categories"=>["Biological Sciences"], "users"=>["Justine E. Alford", "Jade Gumbs", "Emma C. Anderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0091429.g003", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_AP_1_and_AP_3_are_not_required_for_cellular_uptake_of_lipoplexes_/958169", "title"=>"AP-1 and AP-3 are not required for cellular uptake of lipoplexes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-03-11 03:15:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1415343"], "description"=>"<p>HeLa cells were transfected with no siRNA (−, <b>A</b>), siRNAs against AP-1γ, AP-2 µ or AP-3δ (<b>B–D</b> respectively) or a control siRNA (con, <b>E</b>). 24 hours later, the cells were transfected with GFP plasmid using a rhodamine B-labelled lipofection reagent, and fixed after another 24 hours. Rhodamine fluorescence from METAFECTENE®-FluoR (red) is shown in the left panels, GFP fluorescence (green) is shown in the middle panels, and a merged image of the two channels is shown in the right panels. 25 µm scale bars are highlighted in the bottom right of the merged images.</p>", "links"=>[], "tags"=>["biotechnology", "Genetic engineering", "Computational biology", "Molecular genetics", "gene expression", "genetics", "microbiology", "Virology", "Viral transmission and infection", "Viral entry", "Molecular cell biology", "Cellular structures", "cytoplasm", "Membranes and sorting", "Nucleic acids", "ap-3", "localisation"], "article_id"=>958171, "categories"=>["Biological Sciences"], "users"=>["Justine E. Alford", "Jade Gumbs", "Emma C. Anderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0091429.g004", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_AP_1_and_AP_3_are_required_for_nuclear_localisation_of_lipoplexes_/958171", "title"=>"AP-1 and AP-3 are required for nuclear localisation of lipoplexes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-03-11 03:15:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1415344"], "description"=>"<p>Quantitation of the experiments shown in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091429#pone-0091429-g003\" target=\"_blank\">figures 3</a> and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091429#pone-0091429-g004\" target=\"_blank\">4</a>. The percentage of GFP positive cells (<b>A</b>) and METAFECTENE®-FluoR positive cells (<b>B</b>) was counted at 6 hours (grey bars) and 24 hours (black bars) after GFP transfection. For each siRNA treatment at each time point, between five and nine fields of view (600–900 cells) from up to three independent experiments were analysed. Error bars represent the standard error of the mean. Asterisks indicate significant differences to no siRNA (−) samples (<b>A</b>, p = 5.7×10<sup>−5</sup> and 3.1×10<sup>−4</sup> for AP-1 and AP-3 siRNAs at 24 hours respectively; <b>B</b>, p = 0.013 and 0.026 for AP-1 and AP-3 siRNAs at 24 hours respectively).</p>", "links"=>[], "tags"=>["biotechnology", "Genetic engineering", "Computational biology", "Molecular genetics", "gene expression", "genetics", "microbiology", "Virology", "Viral transmission and infection", "Viral entry", "Molecular cell biology", "Cellular structures", "cytoplasm", "Membranes and sorting", "Nucleic acids", "knockdown", "ap-1", "ap-3", "gfp", "fluorescence", "transfected"], "article_id"=>958172, "categories"=>["Biological Sciences"], "users"=>["Justine E. Alford", "Jade Gumbs", "Emma C. Anderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0091429.g005", "stats"=>{"downloads"=>0, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_siRNA_mediated_knockdown_of_AP_1_or_AP_3_reduces_GFP_and_METAFECTENE_174_FluoR_fluorescence_in_transfected_cells_/958172", "title"=>"siRNA-mediated knockdown of AP-1 or AP-3 reduces GFP and METAFECTENE®-FluoR fluorescence in transfected cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-03-11 03:15:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1415347"], "description"=>"<p>HeLa cells were transfected with no siRNA (−), siRNAs against AP-1γ, AP-2 µ or AP-3δ or a control siRNA (con). <b>A.</b> Relative luciferase activity of lysates from cells transfected with <i>Renilla</i> luciferase mRNA 24 hours after siRNAs and harvested after a further 24 hours. The data are from three independent experiments and error bars represent the standard error of the mean. <b>B.</b> GFP fluorescence (green) of cells transfected with GFP mRNA 24 hours after siRNAs and fixed after a further 24 hours. DAPI nuclear stain is shown in blue and 50 µm scale bars are shown in each image. The percentage of GFP positive cells was counted in ten fields of view (1000–2000 cells) for each siRNA treatment, from three independent experiments. The mean percentage of GFP positive cells relative to the no siRNA samples (−), +/− standard error, is shown.</p>", "links"=>[], "tags"=>["biotechnology", "Genetic engineering", "Computational biology", "Molecular genetics", "gene expression", "genetics", "microbiology", "Virology", "Viral transmission and infection", "Viral entry", "Molecular cell biology", "Cellular structures", "cytoplasm", "Membranes and sorting", "Nucleic acids", "ap-3", "transfection"], "article_id"=>958175, "categories"=>["Biological Sciences"], "users"=>["Justine E. Alford", "Jade Gumbs", "Emma C. Anderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0091429.g006", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_AP_1_and_AP_3_are_not_required_for_transfection_with_mRNA_/958175", "title"=>"AP-1 and AP-3 are not required for transfection with mRNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-03-11 03:15:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1415331"], "description"=>"<p>HeLa cells were harvested 24 or 48 hours post-transfection with no siRNA (−), a control siRNA (con) or siRNAs against AP-1γ, AP-2 µ or AP-3δ. <b>A.</b> Western blot of cell lysates harvested 24 hours (left panels) or 48 hours (right panels) post-siRNA transfection, probed with antibodies against AP-1γ (panels 1 and 3, siRNAs from Life Technologies and Dharmacon respectively), AP-2 µ (panel 5, asterisk indicates the band corresponding to AP-2 µ), AP-3δ (panels 7 and 9, siRNAs from Life Technologies or Dharmacon respectively) or γ-tubulin (panels 2, 4, 6, 8, 10 which are controls for the panel above in each case). <b>B.</b> Western blot of cell lysates probed with antibodies against HIV-1 Gag p55 (panels 1, 3, 5) or γ-tubulin (panels 2, 4, 6) when cells were transfected with an HIV-1 proviral plasmid 24 hours after siRNAs (panels 1–4) or co-transfected with proviral plasmid and siRNAs for 48 hours (panels 5 and 6). Panels 3 and 4 are a repeat of the experiment shown in panels 1 and 2, but using different siRNAs to AP-1γ and AP-3δ.</p>", "links"=>[], "tags"=>["biotechnology", "Genetic engineering", "Computational biology", "Molecular genetics", "gene expression", "genetics", "microbiology", "Virology", "Viral transmission and infection", "Viral entry", "Molecular cell biology", "Cellular structures", "cytoplasm", "Membranes and sorting", "Nucleic acids", "knockdown", "ap-1", "ap-3", "hiv-1", "gag"], "article_id"=>958159, "categories"=>["Biological Sciences"], "users"=>["Justine E. Alford", "Jade Gumbs", "Emma C. Anderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0091429.g001", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_siRNA_mediated_knockdown_of_AP_1_or_AP_3_reduces_HIV_1_Gag_gene_expression_/958159", "title"=>"siRNA-mediated knockdown of AP-1 or AP-3 reduces HIV-1 Gag gene expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-03-11 03:15:09"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2014-01-01T00:00:00Z", "end_date"=>"2014-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Genetics", "average_usage"=>[306, 482]}, {"subject_area"=>"/Biology and life sciences/Microbiology", "average_usage"=>[317]}, {"subject_area"=>"/Medicine and health sciences", "average_usage"=>[285]}]}
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