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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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Image restoration by deconvolution. A series of Z slices were obtained from MRC5 cells labelled for RPA (in A) or PCNA (in B) in both confocal and STED modes. The images were then restored using the CMLE deconvolution algorithm of Huygens (SVI). Scale bars = 2 μm.
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Figure 3: Image restoration by deconvolution. A series of Z slices were obtained from MRC5 cells labelled for RPA (in A) or PCNA (in B) in both confocal and STED modes. The images were then restored using the CMLE deconvolution algorithm of Huygens (SVI). Scale bars = 2 μm.

Mentions: Deconvolution algorithms calculate and reposition the parts of the image that are derived from degradation of the light paths due to diffraction within the instrument [32]. We used the Huygens image analysis software from Scientific Volume Imaging to apply deconvolution to the data. We used a theoretical point spread function based on microscope parameters and model ("Confocal" parameter set of Huygens) and the Classic Maximum Likelihood Estimation algorithm to restore the images. High resolution Z stacks of RPA- and PCNA- stained nuclei acquired with the confocal or STED setup were processed in identical ways (figure 3). The post acquisition processing improves the signal to noise ratio in both cases; the images have more distinct foci in each case as the processing removes background blur. As was apparent before deconvolution, the resolution improvement obtained in STED mode reduces the apparent size of the factories containing either RPA (figure 3A) or PCNA (figure 3B), and also the number of individual factories visualised is greater in STED than in confocal mode.


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

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

Image restoration by deconvolution. A series of Z slices were obtained from MRC5 cells labelled for RPA (in A) or PCNA (in B) in both confocal and STED modes. The images were then restored using the CMLE deconvolution algorithm of Huygens (SVI). Scale bars = 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Image restoration by deconvolution. A series of Z slices were obtained from MRC5 cells labelled for RPA (in A) or PCNA (in B) in both confocal and STED modes. The images were then restored using the CMLE deconvolution algorithm of Huygens (SVI). Scale bars = 2 μm.
Mentions: Deconvolution algorithms calculate and reposition the parts of the image that are derived from degradation of the light paths due to diffraction within the instrument [32]. We used the Huygens image analysis software from Scientific Volume Imaging to apply deconvolution to the data. We used a theoretical point spread function based on microscope parameters and model ("Confocal" parameter set of Huygens) and the Classic Maximum Likelihood Estimation algorithm to restore the images. High resolution Z stacks of RPA- and PCNA- stained nuclei acquired with the confocal or STED setup were processed in identical ways (figure 3). The post acquisition processing improves the signal to noise ratio in both cases; the images have more distinct foci in each case as the processing removes background blur. As was apparent before deconvolution, the resolution improvement obtained in STED mode reduces the apparent size of the factories containing either RPA (figure 3A) or PCNA (figure 3B), and also the number of individual factories visualised is greater in STED than in confocal mode.

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