Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.
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 da...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3334904?pdf=render |
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| author | Ronan Broderick Sivaramakrishnan Ramadurai Katalin Tóth Denisio M Togashi Alan G Ryder Jörg Langowski Heinz Peter Nasheuer |
| author_facet | Ronan Broderick Sivaramakrishnan Ramadurai Katalin Tóth Denisio M Togashi Alan G Ryder Jörg Langowski Heinz Peter Nasheuer |
| author_sort | Ronan Broderick |
| collection | DOAJ |
| description | 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. |
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| id | doaj.art-047d8be4478e45f3a39ad306bcde3cf1 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T08:06:21Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-047d8be4478e45f3a39ad306bcde3cf12022-12-21T19:10:46ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0174e3553710.1371/journal.pone.0035537Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.Ronan BroderickSivaramakrishnan RamaduraiKatalin TóthDenisio M TogashiAlan G RyderJörg LangowskiHeinz Peter NasheuerEukaryotic 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.http://europepmc.org/articles/PMC3334904?pdf=render |
| spellingShingle | Ronan Broderick Sivaramakrishnan Ramadurai Katalin Tóth Denisio M Togashi Alan G Ryder Jörg Langowski Heinz Peter Nasheuer Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. PLoS ONE |
| title | Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
| title_full | Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
| title_fullStr | Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
| title_full_unstemmed | Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
| title_short | Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
| title_sort | cell cycle dependent mobility of cdc45 determined in vivo by fluorescence correlation spectroscopy |
| url | http://europepmc.org/articles/PMC3334904?pdf=render |
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