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Analysis of replication factories in human cells by super-resolution light microscopy.

Cseresnyes Z, Schwarz U, Green CM - BMC Cell Biol. (2009)

Bottom Line: The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging.The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported.This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

ABSTRACT

Background: DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution.

Results: Using immunofluorescent imaging of PCNA (proliferating cell nuclear antigen) and RPA (replication protein A) we show that factories are smaller in size (approximately 150 nm diameter), and greater in number (up to 1400 in an early S- phase nucleus), than is determined by confocal imaging. The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging.

Conclusions: These measurements for replication factory size now approach the dimensions suggested by electron microscopy. This agreement between these two methods, that use very different sample preparation and imaging conditions, suggests that we have arrived at a true measurement for the size of these structures. The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported. This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.

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STED imaging of replication factories. MRC5 cells were labelled with ATTO 647-linked secondary antibodies and anti-RPA (in A) or anti-PCNA (in B) primary antibodies. Images were acquired sequentially, in normal confocal mode (green) then using the STED setup (magenta). The lower panels are magnified regions of the cells as indicated. Scale bars = 2 μm.
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Figure 2: STED imaging of replication factories. MRC5 cells were labelled with ATTO 647-linked secondary antibodies and anti-RPA (in A) or anti-PCNA (in B) primary antibodies. Images were acquired sequentially, in normal confocal mode (green) then using the STED setup (magenta). The lower panels are magnified regions of the cells as indicated. Scale bars = 2 μm.

Mentions: To test whether STED microscopy can enhance the visualisation of replication structures we analysed RPA- and PCNA- labelled cells under otherwise identical imaging conditions using the confocal and STED modes on the Leica TCS STED microscope (figure 2). The STED mode utilises a depletion beam at 750 nm that effectively depletes the emission from the Atto 647N dye, resulting in a reduction of the excitation spot and consequent resolution improvement. This is a purely physical method for increasing the resolving power of the microscope - it does not depend on any mathematical processing of the images [28,24,29]. Both the RPA- (figure 2A) and PCNA- (figure 2B) labelled images were dramatically altered by the use of the STED mode. In each case the replication foci appear smaller, sharper and greater in number. This latter change results from the fact that many smaller foci can be resolved in the STED mode, which merged into one continuous structure in the confocal mode due to its limited resolution (figure 2A and 2B, lower magnified panels).


Analysis of replication factories in human cells by super-resolution light microscopy.

Cseresnyes Z, Schwarz U, Green CM - BMC Cell Biol. (2009)

STED imaging of replication factories. MRC5 cells were labelled with ATTO 647-linked secondary antibodies and anti-RPA (in A) or anti-PCNA (in B) primary antibodies. Images were acquired sequentially, in normal confocal mode (green) then using the STED setup (magenta). The lower panels are magnified regions of the cells as indicated. Scale bars = 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803164&req=5

Figure 2: STED imaging of replication factories. MRC5 cells were labelled with ATTO 647-linked secondary antibodies and anti-RPA (in A) or anti-PCNA (in B) primary antibodies. Images were acquired sequentially, in normal confocal mode (green) then using the STED setup (magenta). The lower panels are magnified regions of the cells as indicated. Scale bars = 2 μm.
Mentions: To test whether STED microscopy can enhance the visualisation of replication structures we analysed RPA- and PCNA- labelled cells under otherwise identical imaging conditions using the confocal and STED modes on the Leica TCS STED microscope (figure 2). The STED mode utilises a depletion beam at 750 nm that effectively depletes the emission from the Atto 647N dye, resulting in a reduction of the excitation spot and consequent resolution improvement. This is a purely physical method for increasing the resolving power of the microscope - it does not depend on any mathematical processing of the images [28,24,29]. Both the RPA- (figure 2A) and PCNA- (figure 2B) labelled images were dramatically altered by the use of the STED mode. In each case the replication foci appear smaller, sharper and greater in number. This latter change results from the fact that many smaller foci can be resolved in the STED mode, which merged into one continuous structure in the confocal mode due to its limited resolution (figure 2A and 2B, lower magnified panels).

Bottom Line: The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging.The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported.This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

ABSTRACT

Background: DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution.

Results: Using immunofluorescent imaging of PCNA (proliferating cell nuclear antigen) and RPA (replication protein A) we show that factories are smaller in size (approximately 150 nm diameter), and greater in number (up to 1400 in an early S- phase nucleus), than is determined by confocal imaging. The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging.

Conclusions: These measurements for replication factory size now approach the dimensions suggested by electron microscopy. This agreement between these two methods, that use very different sample preparation and imaging conditions, suggests that we have arrived at a true measurement for the size of these structures. The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported. This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.

Show MeSH