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

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.

Figure 1

RPA and PCNA labelled replication factories. A and B: MRC5 cells were pulse labelled for 10 minutes with 40 μM EdU to label newly synthesised DNA which was then visualised in the green channel alongside indirect immunofluorescence (in magenta) from anti-PCNA or -RPA antibodies respectively. C and D: cells were co-labelled with anti-RPA monoclonal and anti-PCNA polyclonal antibodies in green and magenta (RPA-green in C and PCNA-green in D). Scale bars = 2 μm.

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.

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.

Figure 4

Quantification of replication factory size and number. Three dimensional volume renderings of the restored Z stacks were produced in SVI's Huygens imaging software (panel A - images shown are using a garbage volume of 5 voxels). Scale bars = 2 μm. Colours represent increasing intensities from blue to red. The number of objects in each cell was then counted using a selection of different garbage volume thresholds. The average number of objects per μm³ at each garbage volume (as indicated on the right) is presented (B). The total number of objects for each threshold corresponds to the top of the appropriately shaded bar in each category. These data are also presented in table 1. The same software was used to determine the maximum axial width of an object in each category at garbage volume 5 (C). Error bars represent average deviations from the mean (n = 3-5).

Figure 5

Hydroxyurea treatment affects both replication factory number and size. High resolution Z stacks from cells treated with hydroxyurea, or untreated, were processed as for figure 4 (A). The average maximum axial width (B) and number of objects (C) are shown, calculated exactly as in figure 3.