Visualisation of PCNA monoubiquitination in vivo by single pass spectral imaging FRET microscopy.

PloS One
Christopher BattersJulian E Sale

Abstract

Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination in both fixed and live cells with a single imaging pass. We validate the method with genetic controls in the avian cell line DT40 and use it to examine the intracellular dynamics of PCNA ubiquitination following subnuclear UV irradiation. This general approach is likely to be of utility for live imaging of post-translational modifications of a wide range of substrates in vivo.

References

Jun 13, 2002·Proceedings of the National Academy of Sciences of the United States of America·Robert E CampbellRoger Y Tsien
Dec 28, 2002·Cell Structure and Function·Yasushi HiraokaTokuko Haraguchi
Nov 5, 2003·Nature Biotechnology·Elizabeth A Jares-Erijman, Thomas M Jovin
Mar 23, 2005·Nature Methods·Julie PerroyMichel Bouvier
Oct 18, 2005·Molecular and Cellular Biology·Jeroen EssersWim Vermeulen
Mar 15, 2006·Nature Cell Biology·Tony T HuangAlan D D'Andrea
Mar 16, 2006·Proceedings of the National Academy of Sciences of the United States of America·Sundar GanesanFred S Wouters
Apr 28, 2006·Molecular Biology of the Cell·Jonathan FramptonAlan R Lehmann
Nov 16, 2006·PLoS Biology·Hiroshi ArakawaJean-Marie Buerstedde
Nov 17, 2006·Proceedings of the National Academy of Sciences of the United States of America·Ildiko UnkLajos Haracska
Dec 18, 2007·Molecular Cell·Vibe H OestergaardKetan J Patel
Mar 5, 2008·Proceedings of the National Academy of Sciences of the United States of America·Ildiko UnkLajos Haracska
Aug 23, 2008·Proceedings of the National Academy of Sciences of the United States of America·Akira MotegiKyungjae Myung
Sep 12, 2008·Microscopy Research and Technique·Diego MegíasMaría C Montoya
Oct 11, 2008·Proceedings of the National Academy of Sciences of the United States of America·Atsuko NiimiAlan R Lehmann
Oct 25, 2008·Cell Cycle·Sufang ZhangMarietta Y W T Lee

❮ Previous
Next ❯

Citations

Jan 1, 2014·Proceedings of the National Academy of Sciences of the United States of America·Christopher BattersClaudia Veigel
Nov 19, 2010·Journal of Cell Science·Micha Nethe, Peter L Hordijk

❮ Previous
Next ❯

Methods Mentioned

BETA
ubiquitination
fluorescence microscopy
FRET
BRET

Software Mentioned

BRET
Nikon EZ - C1

Related Concepts

Related Feeds

Bacterial Cell Wall Structure (ASM)

Bacterial cell walls are made of peptidoglycan (also called murein), which is made from polysaccharide chains cross-linked by unusual peptides containing D-amino acids. Here is the latest research on bacterial cell wall structures.

Bacterial Cell Wall Structure

Bacterial cell walls are made of peptidoglycan (also called murein), which is made from polysaccharide chains cross-linked by unusual peptides containing D-amino acids. Here is the latest research on bacterial cell wall structures.