Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy
Broderick, Ronan Ramadurai, Sivaramakrishnan Tóth, Katalin Togashi, Denisio M. Ryder, Alan G. Langowski, Jörg Nasheuer, Heinz-Peter
Broderick, Ronan
Ramadurai, Sivaramakrishnan
Tóth, Katalin
Togashi, Denisio M.
Ryder, Alan G.
Langowski, Jörg
Nasheuer, Heinz-Peter
Loading...
Repository DOI
Publication Date
2012-04-19
Type
Article
Downloads
Citation
Broderick R, Ramadurai S, Tóth K, Togashi DM, Ryder AG, Langowski J, et al. (2012) Cell Cycle-Dependent Mobility of Cdc45 Determined in vivo by Fluorescence Correlation Spectroscopy. PLoS ONE 7(4): e35537. https://doi.org/10.1371/journal.pone.0035537
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.
Funder
Publisher
Public Library of Science
Publisher DOI
10.1371/journal.pone.0035537
Rights
Attribution-NonCommercial-NoDerivs 3.0 Ireland