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

Visualizing helicases unwinding DNA at the single molecule level

DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes

Adaptive Traits Are Maintained on Steep Selective Gradients despite Gene Flow and Hybridization in the Intertidal Zone

Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia) population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus (from Northern Portugal) was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients acts to maintain the 3 distinct genetic and morphological taxa within their preferred vertical distribution ranges. On the strength of distributional, genetic, physiological and morphological differences, we propose elevation of F. spiralis var. platycarpus from variety to species level, as F. guiryi

Detection of PCNA ubiquitination by acceptor photobleaching FRET.

<p>A. A <i>usp1</i> DT40 cell before and after bleaching of the mRFP acceptor. B. Whole cell spectrum, of the cell shown in A, with excitation at 515 nm (Ex515/ΣλmRFP) before (black line) and after (grey line) photobleaching. C. Whole cell spectrum with excitation at 407 nm (Ex407/ΣλCFP) before and after photobleaching. Inset panel shows a zoomed in region covering the wavelengths around the mRFP emission maximum at 607 nm. D. Ensemble averages from 30 cells of the indicated genotype before and after photobleaching of mRFP. Intensity was normalised to pre-photobleach maximum.</p

FRET between CFP and mRFP in solution.

<p>A. Absorbance and emission spectra of CFP and mRFP. The spectra are normalised to the excitation or emission maximum. The overlap (Jλ) between CFP emission and mRFP excitation is shown in green. The overlap in the emission spectra is shown in black. B. Schematic of the CFP-mRFP fusion protein. Before cleavage with TEV protease FRET can take place between CFP and mRFP resulting in energy from the excited CFP being transferred to the mRFP, diminishing the direct emission by CFP at 478 nm and stimulating emission of RFP at 607 nm. Following cleavage, energy transfer is lost resulting in an increase in 478 nm emission by CFP. C. Cleavage of the mRFP-TEV-CFP fusion protein by TEV protease. An aliquot of the reaction was taken from the spectrophotometer at the point at which the CFP emission peak had plateaued following addition of TEV protease. Aliquots were run on SDS-PAGE, stained with Coomassie Blue and transferred for Western blotting with anti-CFP and anti-RFP. D. FRET between CFP and mRFP. Spectra of the CFP-TEV-mRFP fusion protein before (red line) and after (green line) cleavage with TEV protease. The purple line shows the blank control and blue line the emission of the mRFP component of the fusion protein when directly excited at 584 nm.</p

Expression of fluorescently tagged PCNA and ubiquitin in DT40 cells.

<p>A. The genetics of PCNA monoubiquitination. PCNA is monoubiquitinated at lysine 164 by RAD6/RAD18. The monoubiquitin can be cleaved from PCNA by USP1. Thus, when ubiquitin is conjugated to PCNA, FRET may take place between the CFP attached to ubiquitin and mRFP attached to PCNA. No FRET should be seen in a <i>pcna</i>K164R cell line and the signal should be exaggerated in a <i>usp1</i> line. B & C Expression of CFP-ubiquitin and mRFP-PCNA in DT40. B. FACS plots. C. Western blots.</p

Detection of PCNA ubiquitination <i>in vivo</i> by spectral imaging FRET.

<p>A. Reference spectra acquired with the Nikon C1-si for CFP (Ex407/ΣλCFP, blue line), mRFP (Ex515/ΣλmRFP, red line) and autofluorescence (‘Junk’) generated by excitation with the 407 nm laser (green line). The dip in the CFP and ‘Junk’ spectra at 515 nm are due to the metal finger protecting the PMT array from the 515 nm laser line. B. Correction of CFP spectral bleedthrough by spectral unmixing. The X axis plots the intensities of the CFP spectral signal (ΣλCFP) following excitation with 407 nm light from whole cells against the Y axis which plots the corresponding mRFP bleedthrough signal, ⁴⁰⁷ΣλmRFP. C. Direct excitation of mRFP by 407 nm light. The X axis plots the intensities of the CFP spectral signal (ΣλCFP) following excitation with 407 nm light from whole cells against the Y axis which plots the corresponding mRFP signal, ΣλmRFP. D. Example of unmixed spectral images of DT40 taken with 407 nm light (left hand panel) and 515 nm light (right hand panel) 20 minutes after UV irradiation through a 3 µm microporous filter. In the left hand image, the unmixed CFP (⁴⁰⁷ΣλCFP) and mRFP (⁴⁰⁷ΣλmRFP) channels are superimposed and ⁴⁰⁷ΣλmRFP enhanced to allow it to be seen in the merge i.e. this image depicts the position of the two signals but not their absolute intensities. E. Uncorrected FRET ratio image (⁴⁰⁷ΣλmRFP∶⁴⁰⁷ΣλCFP). LUT  =  look-up table. F. FRET ratio image determined after pixel-by-pixel correction for direct excitation of mRFP by the 407 nm laser. G. FRET ratio with the threshold applied H. Derivation of the FRET threshold. Scatter plot of ΣλCFP when excited with 407 nm light against the raw FRET ratio derived from the algorithm in D applied to a <i>usp1</i> cell field (red) and a <i>pcna</i>K164R cell field (blue). The dotted line (at a ratio of 0.1) shows the upper cut off for 99% of the pixels for the <i>pcna</i>K164R cell. I. An example of a cell with a focal accumulation of PCNA that does not result in a FRET signal demonstrating the independence of the FRET signal from mRFP bleedthrough. See also legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009008#pone-0009008-g005" target="_blank">Figure 5D</a>.</p

Direct visualisation of PCNA ubiquitination by spectral imaging of fixed DT40 cells.

<p>A. Example images of cell mixtures with regions of interest, red circle  =  cell expressing mRFP-PCNA only, blue circle  =  CFP-ubiquitin only and green circle  =  both. B. Ensemble average spectra from cells expressing mRFP-PCNA and/or CFP-ubiquitin. Key as above. The emission maximum of mRFP is 607 nm. C. Relationship of directly excited intensity of a fluorophore to its potential, indirect, contribution to the FRET signal (see text).</p