Visualisation of PCNA monoubiquitination in vivo by single pass spectral imaging FRET microscopy.
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.
Batters
Christopher
C
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Zhu
Hannah
H
Sale
Julian E
JE
D000818:Animals; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism / Q000528:radiation effects; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D054875:Ubiquitination; D014466:Ultraviolet Rays; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein