Knockdown of SCFSkp2 Function Causes Double-Parked Accumulation in the Nucleus and DNA Re-Replication in Drosophila Plasmatocytes
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{"title"=>"Knockdown of SCFSkp2 Function Causes Double-Parked Accumulation in the Nucleus and DNA Re-Replication in Drosophila Plasmatocytes", "type"=>"journal", "authors"=>[{"first_name"=>"Paul T.", "last_name"=>"Kroeger", "scopus_author_id"=>"53463857300"}, {"first_name"=>"Douglas A.", "last_name"=>"Shoue", "scopus_author_id"=>"6507369998"}, {"first_name"=>"Frank M.", "last_name"=>"Mezzacappa", "scopus_author_id"=>"55899364300"}, {"first_name"=>"Gary F.", "last_name"=>"Gerlach", "scopus_author_id"=>"55191562500"}, {"first_name"=>"Rebecca A.", "last_name"=>"Wingert", "scopus_author_id"=>"8750982000"}, {"first_name"=>"Robert A.", "last_name"=>"Schulz", "scopus_author_id"=>"36459131800"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24205363", "sgr"=>"84886257655", "doi"=>"10.1371/journal.pone.0079019", "scopus"=>"2-s2.0-84886257655", "pui"=>"370104552", "isbn"=>"1932-6203", "issn"=>"19326203"}, "id"=>"b5ece879-9a9a-309f-9ca5-781084595697", "abstract"=>"In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF(Skp2) complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type.", "link"=>"http://www.mendeley.com/research/knockdown-scfskp2-function-causes-doubleparked-accumulation-nucleus-dna-rereplication-drosophila-pla", "reader_count"=>14, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>3, "Student > Master"=>2, "Student > Bachelor"=>3}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>3, "Student > Master"=>2, "Student > Bachelor"=>3}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>9, "Immunology and Microbiology"=>1, "Medicine and Dentistry"=>1, "Business, Management and Accounting"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>9}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1257572", "https://ndownloader.figshare.com/files/1257573", "https://ndownloader.figshare.com/files/1257574"], "description"=>"<div><p>In <i>Drosophila</i>, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. <i>eater-Gal4</i>;<i>UAS-GFP</i> flies were crossed to <i>UAS-RNAi</i> lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF<sup>Skp2</sup> complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type.</p> </div>", "links"=>[], "tags"=>["knockdown", "causes", "double-parked", "accumulation", "nucleus", "dna", "re-replication", "plasmatocytes"], "article_id"=>832045, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0079019.s001", "https://dx.doi.org/10.1371/journal.pone.0079019.s002", "https://dx.doi.org/10.1371/journal.pone.0079019.s003"], "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Knockdown_of_SCF_Skp2_Function_Causes_Double_Parked_Accumulation_in_the_Nucleus_and_DNA_Re_Replication_in_Drosophila_Plasmatocytes/832045", "title"=>"Knockdown of SCF<sup>Skp2</sup> Function Causes Double-Parked Accumulation in the Nucleus and DNA Re-Replication in <i>Drosophila</i> Plasmatocytes", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-10-24 03:26:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1257568"], "description"=>"<p>(A-C) <i>pxnGal4>w</i><sup><i>1118</i></sup> control hemolymph samples stained with (A) α-Cyclin E, (B) α-Gem, and (C) α-Dup and (D-F) DAPI. (G-I) <i>pxnGal4>UAS-lin19 </i><i>RNAi</i><sup><i>HM05197</i></sup> hemocytes stained with (G) α-Cyclin E, (H) α-Gem, and (I) α-Dup and (J-L) DAPI. (M-O) <i>pxnGal4> UAS-Gem</i><sup><i>43</i></sup><i>; UAS-lin19 </i><i>RNAi</i><sup><i>HM05197</i></sup> hemocytes stained with (M) α-Cyclin E, (N) α-Gem, and (O) α-Dup and (P-R) DAPI. (S) Graph indicating the number of cells that fall into specific size categories for <i>pxnGal4>w</i><sup><i>1118</i></sup>, <i>pxnGal4>UAS-Gem</i><sup>43</sup>, <i>pxnGal4>UAS-lin19 </i><i>RNAi</i><sup><i>HM05197</i></sup>, and <i>pxnGal4>UAS-Gem</i><sup><i>43</i></sup><i>; UAS-lin19 </i><i>RNAi</i><sup><i>HM05197</i></sup> genotypes. As indicated by the graph, overexpression of Gem in the SCF knockdown background causes a great reduction in the number of giant cells when compared to knockdown of the SCF complex only. Nuclear stained cells indicated by white filled arrows, cytoplasmic stained cells indicated by outlined arrows. Scale bar = 20 μm.</p>", "links"=>[], "tags"=>["gem", "elicits", "scf", "knockdown", "plasmatocyte"], "article_id"=>832041, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079019.g006", "stats"=>{"downloads"=>1, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overexpression_of_Gem_elicits_a_partial_rescue_of_SCF_knockdown_plasmatocyte_phenotypes_/832041", "title"=>"Overexpression of Gem elicits a partial rescue of SCF knockdown plasmatocyte phenotypes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-24 03:26:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1257567"], "description"=>"<p>(A-C) <i>pxnGal4>w</i><sup><i>1118</i></sup> control hemolymph samples stained with (A) α-Cyclin E, (B) α-Gem, and (C) α-Dup and DAPI (A’-C’). (D-F) <i>pxnGal4>UAS-lin19</i><sup><i>HM05197</i></sup> stained with (D) α-Cyclin E, (E) α-Gem, and (F) α-Dup and DAPI (D’-F’). (G-R) <i>pxnGal4>UAS-SCF </i><i>complex </i><i>member </i><i>RNAi</i> hemocytes stained with (Column 1) α-Cyclin E, (Column 2) α-Gem, and (Column 3) α-Dup. (S-U) <i>pxnGal4>UAS-Cyclin </i><i>E </i><i>RNAi</i><sup><i>JF02473</i></sup> hemocytes stained with (S) α-Cyclin E, (T) α-Gem, and (U) α-Dup. Nuclear stained enlarged cells are indicated by white filled arrows cytoplasmic stained enlarged cells are indicated by outlined arrows. Scale bar = 20 μm.</p>", "links"=>[], "tags"=>["scf", "members", "causes", "subcellular", "localization"], "article_id"=>832040, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079019.g005", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knockdown_of_SCF_complex_members_causes_changes_in_subcellular_localization_of_cell_cycle_regulatory_proteins_/832040", "title"=>"Knockdown of SCF complex members causes changes in subcellular localization of cell cycle regulatory proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-24 03:26:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1257562"], "description"=>"<p>(A,B) <i>PxnGal4>w</i><sup><i>1118</i></sup> wild-type animals were fed BrdU during larval stages resulting in slight incorporation into the DNA as visualized by anti-BrdU and DAPI staining. (C,D) <i>pxnGal4>lin19 </i><i>RNAi</i><sup><i>HM05197</i></sup> were fed BrdU during laval stages resulting in a distinct nuclear BrdU staining in the nucleus when observed with DAPI staining. Scale bar = 20 μm.</p>", "links"=>[], "tags"=>["cells", "elicited", "knockdown", "brdu"], "article_id"=>832038, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079019.g004", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Enlarged_cells_elicited_by_functional_knockdown_of_lin19_exhibit_increased_nuclear_BrdU_staining_/832038", "title"=>"Enlarged cells elicited by functional knockdown of <i>lin19</i> exhibit increased nuclear BrdU staining.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-24 03:26:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1257559"], "description"=>"<p>(A) <i>eaterGal4>UAS-GFP</i> is present in control larval circulating hemocytes. (B) <i>eaterGal4>UAS-GFP>lin19 </i><i>RNAi</i><sup><i>HM05197</i></sup> hemolymph samples show GFP-positive enlarged cells, indicated by the arrow. (C) Cullin-1 (<i>Drosophila</i> homolog is Lin19) belongs to the SCF ubiquitin ligase complex. The SCF complex is inhibited by Cand1 binding to Cullin. To activate the complex, Cand1 is displaced by Nedd8 allowing the Skp/F-box protein pair to bind Cullin giving specificity to the complex to ubiquitinate the target substrate. Scale bars = 20 μm.</p>", "links"=>[], "tags"=>["scf", "enlarged", "plasmatocyte"], "article_id"=>832035, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079019.g001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loss_of_function_of_lin19_a_member_of_the_SCF_complex_results_in_an_enlarged_plasmatocyte_phenotype_/832035", "title"=>"Loss-of-function of <i>lin19</i>, a member of the SCF complex, results in an enlarged plasmatocyte phenotype.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-24 03:26:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1257570"], "description"=>"<p>The SCF complex that functions in hematopoiesis consists of Lin19, SkpA, Skp2, Roc1a and Nedd8. This complex functions to ubiquitinate its substrate, Dup, which is necessary for the proper cell cycle progression of plasmatocytes.</p>", "links"=>[], "tags"=>["scf", "plasmatocyte"], "article_id"=>832043, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079019.g007", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Model_of_the_SCF_complex_involved_in_Drosophila_plasmatocyte_development_/832043", "title"=>"Model of the SCF complex involved in <i>Drosophila</i> plasmatocyte development.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-24 03:26:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1257561"], "description"=>"<p>(A-D) <i>pxnGal4>w</i><sup><i>1118</i></sup> control hemolymph samples stained with (A) DAPI, (B) α-acetylated-Tubulin, (C) α-gamma-Tubulin, and (D) merge. (E-H) <i>pxnGal4>UAS-gem </i><i>RNAi</i><sup><i>M4</i></sup> positive re-replication control hemolymph samples were stained with (E) DAPI, (F) α-acetylated-Tubulin, (G) α-gamma-Tubulin, and (H) merge. (I-L) <i>pxnGal4>UAS-lin19 </i><i>RNAi</i><sup><i>HM05197</i></sup> hemocytes were stained with (I) DAPI, (J) α-acetylated-Tubulin, (K) α-gamma-Tubulin, and (L) merge. Inserts in C, G, and K are a 100% α-gamma-Tubulin magnification. Scale bar = 10 μm.</p>", "links"=>[], "tags"=>["over-replicated", "dna", "centrioles", "formed", "knockdown", "scf"], "article_id"=>832037, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079019.g003", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cells_with_over_replicated_DNA_and_multiple_centrioles_are_formed_after_knockdown_of_SCF_complex_member_lin19_/832037", "title"=>"Cells with over-replicated DNA and multiple centrioles are formed after knockdown of SCF complex member <i>lin19</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-24 03:26:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1257560"], "description"=>"<div><p>(A) Control <i>w</i><sup>1118</sup> circulating plasmatocytes are marked by an <i>eaterGal4>UAS-GFP</i> transgene. (C,E,G,I,K) <i>eater>GFP>UAS-gene </i><i>RNAi</i> expressed in circulating hemocytes causes enlarged GFP-positive cells indicated by arrows. (B) <i>pxn</i>><i>w</i><sup>1118</sup> control hemocytes stained with anti-P1, a plasmatocyte specific antibody. (D,F,H,J,L) <i>pxn</i>>UAS<i>-gene </i><i>RNAi</i> hemolymph samples stained with anti-P1 detects enlarged cells labeled with the plasmatocyte-specific antibody indicated by arrows. </p>\n\t\t\t\t\t\t<p>Scale bars = 20 μm.</p></div>", "links"=>[], "tags"=>["enlarged", "plasmatocyte", "phenotype", "elicited", "rnai", "knockdown", "members", "scf"], "article_id"=>832036, "categories"=>["Biological Sciences"], "users"=>["Paul T. Kroeger Jr.", "Douglas A. Shoue", "Frank M. Mezzacappa", "Gary F. Gerlach", "Rebecca A. Wingert", "Robert A. Schulz"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079019.g002", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_An_enlarged_plasmatocyte_phenotype_elicited_by_RNAi_knockdown_of_members_of_a_specific_SCF_complex_/832036", "title"=>"An enlarged plasmatocyte phenotype elicited by RNAi knockdown of members of a specific SCF complex.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-24 03:26:59"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Biology and life sciences/Cell biology", "average_usage"=>[272, 472, 600, 713, 815, 911, 1004, 1094, 1185, 1273, 1358, 1441]}, {"subject_area"=>"/Medicine and health sciences", "average_usage"=>[264, 460, 584, 692, 794, 887, 978, 1067, 1154, 1241, 1328, 1408, 1474]}, {"subject_area"=>"/Medicine and health sciences/Immunology", "average_usage"=>[262, 463, 587, 693, 790, 882, 965, 1051, 1135, 1218, 1300, 1376, 1438]}]}
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