Cell Cycle-Dependent Mobility of Cdc45 Determined in vivo by Fluorescence Correlation Spectroscopy
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{"title"=>"Cell cycle-dependent mobility of Cdc45 determined in vivo by Fluorescence Correlation Spectroscopy", "type"=>"journal", "authors"=>[{"first_name"=>"Ronan", "last_name"=>"Broderick", "scopus_author_id"=>"57197551233"}, {"first_name"=>"Sivaramakrishnan", "last_name"=>"Ramadurai", "scopus_author_id"=>"35316363100"}, {"first_name"=>"Katalin", "last_name"=>"Tóth", "scopus_author_id"=>"56470919600"}, {"first_name"=>"Denisio M.", "last_name"=>"Togashi", "scopus_author_id"=>"6603127093"}, {"first_name"=>"Alan G.", "last_name"=>"Ryder", "scopus_author_id"=>"7005264150"}, {"first_name"=>"Jörg", "last_name"=>"Langowski", "scopus_author_id"=>"7005737357"}, {"first_name"=>"Heinz Peter", "last_name"=>"Nasheuer", "scopus_author_id"=>"7004573323"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"22536402", "sgr"=>"84859856402", "doi"=>"10.1371/journal.pone.0035537", "scopus"=>"2-s2.0-84859856402", "pui"=>"364639765", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"19326203"}, "id"=>"6b4b14c9-d430-3795-a330-c5dac4f97757", "abstract"=>"Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.", "link"=>"http://www.mendeley.com/research/cell-cycledependent-mobility-cdc45-determined-vivo-fluorescence-correlation-spectroscopy", "reader_count"=>27, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>4, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>8, "Student > Master"=>2, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>4}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>4, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>8, "Student > Master"=>2, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>4}, "reader_count_by_subject_area"=>{"Engineering"=>3, "Biochemistry, Genetics and Molecular Biology"=>5, "Materials Science"=>1, "Agricultural and Biological Sciences"=>13, "Medicine and Dentistry"=>1, "Physics and Astronomy"=>1, "Chemistry"=>2, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>3}, "Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>2}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>13}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}}, "reader_count_by_country"=>{"United States"=>1, "Ireland"=>1, "Germany"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/337992", "https://ndownloader.figshare.com/files/338067", "https://ndownloader.figshare.com/files/338146", "https://ndownloader.figshare.com/files/338195", "https://ndownloader.figshare.com/files/338243", "https://ndownloader.figshare.com/files/338292"], "description"=>"<div><p>Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) <em>in vivo</em> in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the <em>in vivo</em> FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.</p> </div>", "links"=>[], "tags"=>["cycle-dependent", "mobility", "cdc45", "determined", "fluorescence", "spectroscopy"], "article_id"=>126797, "categories"=>["Physics", "Biochemistry", "Cell Biology", "Biophysics"], "users"=>["Ronan Broderick", "Sivaramakrishnan Ramadurai", "Katalin Tóth", "Denisio M. Togashi", "Alan G. Ryder", "Jörg Langowski", "Heinz Peter Nasheuer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0035537.s001", "https://dx.doi.org/10.1371/journal.pone.0035537.s002", "https://dx.doi.org/10.1371/journal.pone.0035537.s003", "https://dx.doi.org/10.1371/journal.pone.0035537.s004", "https://dx.doi.org/10.1371/journal.pone.0035537.s005", "https://dx.doi.org/10.1371/journal.pone.0035537.s006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Cell_Cycle_Dependent_Mobility_of_Cdc45_Determined_in_vivo_by_Fluorescence_Correlation_Spectroscopy/126797", "title"=>"Cell Cycle-Dependent Mobility of Cdc45 Determined <em>in vivo</em> by Fluorescence Correlation Spectroscopy", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-04-19 01:53:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/650532"], "description"=>"<p>Panel a, schematic diagram of eGFP-Cdc45 protein encoded by Cdc45L ORF cloned into pIC113gw vector. Panel b, total cell extract HeLaS3 cells (-ve) and Hela S3 cells stably expressing eGFP-Cdc45 (eGFP-Cdc45) normalized for protein content and analysed by western blotting using antibodies raised against Cdc45 and β-Actin, which serves as a loading control. Panel c, western blot analysis of immunoprecipitation of eGFP-Cdc45 using GFP-Trap IP from HeLa S3 cells transiently expressing eGFP-Cdc45. Verification of purification of eGFP-Cdc45 and co-immunoprecipitation of Mcm7 was carried out using antibodies raised against Mcm7 and Cdc45. Input (L), unbound (FT), mock IP (-ve) and IP from cells expressing eGFP-Cdc45 (eGFP) indicate yield of the IP and co-immunoprecipiation. Antibody light chain acts as a loading control.</p>", "links"=>[], "tags"=>["s3", "cells", "stably", "expressing"], "article_id"=>321020, "categories"=>["Physics", "Biochemistry", "Cell Biology", "Biophysics"], "users"=>["Ronan Broderick", "Sivaramakrishnan Ramadurai", "Katalin Tóth", "Denisio M. Togashi", "Alan G. Ryder", "Jörg Langowski", "Heinz Peter Nasheuer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0035537.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_HeLa_S3_cells_stably_expressing_eGFP_Cdc45_/321020", "title"=>"HeLa S3 cells stably expressing eGFP-Cdc45.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-19 00:17:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/650639"], "description"=>"<p>Panel a, chromatin-associated lysate from 1×10<sup>6</sup> Hela S3 cells synchronized at various cell cycle stages by two consecutive thymidine block analysed by western blotting using antibodies raised against Cdc45, Mcm7, Mcm5, Lamin B1, P261 and P125 of Pol ε and <i>δ</i>, respectively. The latter serves as a loading control. Asynchronous control cells (Asn) or cells analysed at times ranging from 0 to 12 h following release from the second thymidine block (TdR 0 to TdR 12) were analysed in parallel by FACS. Corresponding FACS profiles for relevant timepoints are also shown. Panel b, western blot of chromatin-associated Cdc45 following UVC treatment. HeLa S3 cells treated with 5 J/m<sup>2</sup> UVC harvested at indicated timepoints post treatment with untreated cells (UT) acting as a control. Chromatin-associated lysates normalized for protein content were analysed by western blotting using antibodies raised against Cdc45 and Lamin B1, which serves as a loading control.</p>", "links"=>[], "tags"=>["cdc45", "chromatin", "synchronized", "hela", "s3", "cells", "dna"], "article_id"=>321122, "categories"=>["Physics", "Biochemistry", "Cell Biology", "Biophysics"], "users"=>["Ronan Broderick", "Sivaramakrishnan Ramadurai", "Katalin Tóth", "Denisio M. Togashi", "Alan G. Ryder", "Jörg Langowski", "Heinz Peter Nasheuer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0035537.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Association_of_Cdc45_with_chromatin_synchronized_HeLa_S3_cells_and_after_DNA_damage_/321122", "title"=>"Association of Cdc45 with chromatin synchronized HeLa S3 cells and after DNA damage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-19 00:18:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/650753"], "description"=>"<p>The figure shows typical auto-correlation curves of eGFP-Cdc45 (□) in asynchronous HeLa S3 cells stably expressing eGFP-Cdc45. In the upper panel the solid black line corresponds to a two-component free diffusion model and in the lower panel the gray line is the residual of the fit.</p>", "links"=>[], "tags"=>["curves"], "article_id"=>321241, "categories"=>["Physics", "Biochemistry", "Cell Biology", "Biophysics"], "users"=>["Ronan Broderick", "Sivaramakrishnan Ramadurai", "Katalin Tóth", "Denisio M. Togashi", "Alan G. Ryder", "Jörg Langowski", "Heinz Peter Nasheuer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0035537.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Auto_correlation_curves_of_eGFP_Cdc45_/321241", "title"=>"Auto-correlation curves of eGFP-Cdc45.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-19 00:20:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/650906"], "description"=>"<p>Asynchronous (Asn) UVC-treated (+UVC, 5 J/m<sup>2</sup>, 1 h post-treatment), G1/S transition or S phase synchronized HeLa S3 cells stably expressing eGFP-Cdc45 were lysed and normalized for protein content and separated by gel filtration chromatography analysed by western blotting using antibodies raised against Cdc45, Mcm5 and RPA 32. (panels a, b, c, and d, respectively). Theoretical molecular weight (kDa) and Stoke's radius (Å) of protein standards are overlayed. RPA32 acts as a marker for DNA damage response following UVC treatment. FACS analysis is provided for asynchronous (Asn), G1/S transition and S phase synchronized cells (e) and asynchronous cells treated with 5 J/m<sup>2</sup>, 1 h post-treatment (f).</p>", "links"=>[], "tags"=>["cdc45-containing", "complexes", "uv"], "article_id"=>321393, "categories"=>["Physics", "Biochemistry", "Cell Biology", "Biophysics"], "users"=>["Ronan Broderick", "Sivaramakrishnan Ramadurai", "Katalin Tóth", "Denisio M. Togashi", "Alan G. Ryder", "Jörg Langowski", "Heinz Peter Nasheuer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0035537.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_the_size_distribution_of_Cdc45_containing_protein_complexes_during_the_cell_cycle_and_after_UV_damage_/321393", "title"=>"Analysis of the size distribution of Cdc45-containing protein complexes during the cell cycle and after UV damage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-19 00:23:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/651015"], "description"=>"<p>Mean and standard deviations obtained from at least 15 cells.</p>", "links"=>[], "tags"=>["coefficient", "egfp-cdc45", "g1", "uv"], "article_id"=>321497, "categories"=>["Physics", "Biochemistry", "Cell Biology", "Biophysics"], "users"=>["Ronan Broderick", "Sivaramakrishnan Ramadurai", "Katalin Tóth", "Denisio M. Togashi", "Alan G. Ryder", "Jörg Langowski", "Heinz Peter Nasheuer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0035537.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Diffusion_coefficient_of_eGFP_Cdc45_at_G1_to_S_phase_transition_and_in_S_phase_and_after_UV_damage_/321497", "title"=>"Diffusion coefficient of eGFP-Cdc45 at G1 to S phase transition and in S phase and after UV damage.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-04-19 00:24:57"}

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Relative Metric

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