PCNA appears in two populations of slow and fast diffusion with a constant ratio throughout S-phase in replicating mammalian cells - PubMed (original) (raw)

PCNA appears in two populations of slow and fast diffusion with a constant ratio throughout S-phase in replicating mammalian cells

Patrick J M Zessin et al. Sci Rep. 2016.

Abstract

DNA replication is a fundamental cellular process that precedes cell division. Proliferating cell nuclear antigen (PCNA) is a central scaffold protein that orchestrates DNA replication by recruiting many factors essential for the replication machinery. We studied the mobility of PCNA in live mammalian cells using single-particle tracking in combination with photoactivated-localization microscopy (sptPALM) and found two populations. The first population which is only present in cells with active DNA replication, showed slow diffusion and was found to be located in replication foci. The second population showed fast diffusion, and represents the nucleoplasmic pool of unbound PCNA not involved in DNA replication. The ratio of these two populations remained constant throughout different stages of S-phase. A fraction of molecules in both populations showed spatially constrained mobility. We determined an exploration radius of ~100 nm for 13% of the slow-diffusing PCNA molecules, and of ~600 nm for 46% of the fast-diffusing PCNA molecules.

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Figures

Figure 1

Figure 1. Single-molecule tracking of mEos2-PCNA in live replicating HeLa cells.

(A) Cells stably expressing mEos2-PCNA were manually screened for replication foci pattern using the green fluorescence emission of mEos2 (top-right). Tracking of PCNA in living cells reveal different modes of PCNA mobility (top-left) (scale 2 μm). Two exemplary tracks (Dgreen: 2.1 μm2/s; Dblue: 0.0015 μm2/s) are shown in the inset (scale 500 nm). (B) Histogram of the trajectory length of individual PCNA molecules tracked for at least 160 ms (8 frames) (data from 18 cells, 7435 trajectories).

Figure 2

Figure 2. Diffusion coefficient distribution of mEos2-PCNA in replicating and not replicating HeLa (stable cell line).

(A) Two populations of PCNA can be distinguished by their diffusion coefficient. The larger population of PCNA exhibits a low diffusion coefficient (peak 0.02 μm2/s) in cells showing typical patterns of replication (S-phase). In cells without replication foci (non-S-phase) the majority of PCNA molecules exhibit a high diffusion coefficient (peak 1.5 μm2/s) (cells analyzed: 6 fixed cells (3411 tracks); 18 cells with replication foci (7435 tracks); 3 cells without replication foci (554 tracks)). (B) Diffusion coefficient distribution of mEos2-PCNA in cells without replication patterns (non S-phase) and cells treated with 10 μg/ml aphidicolin to stop DNA replication. For comparison, cells were transfected with mEos2 fused to a nuclear localization sequence (NLS) confirming the unbound status of PCNA molecules with D > 0.1 μm2/s (3 cells without replication foci, 554 tracks; 5 cells treated with aphidicolin, 4526 tracks; 5 cells transfected with mEos2-NLS, 135 tracks). (C) Diffusion coefficient distributions for cells in early, mid and late S-phase exhibit a high similarity of the ratios of the slow and fast PCNA population (cells analyzed: 8 cells in early S-phase (3493 tracks); 7 in mid S-phase (3295 tracks); 3 in late S-phase (647 tracks)) (error bars represent s.e.m.).

Figure 3

Figure 3. PCNA molecules are not only distinguishable by diffusion coefficient, but also by radius of confinement.

(A) Both slow and fast PCNA populations exhibit confined (dark grey bars) and free (light grey bars) diffusion. 13% (658 out of 5254 trajectories) of PCNA molecules belonging to the slow-diffusing population (D < 0.1 μm2/s) and 46% (1004 out of 2181 trajectories) belonging to the fast-diffusing population (D > 0.1 μm2/s) exhibit confined diffusion. The radius of confinement was calculated for each single trajectory, and 2D-histogrammed with the diffusion coefficient. PCNA molecules of the slow population show a peak at r = 0.1 μm, whereas the fast population peaks at r = 0.6 μm (total number of tracks: 1662). (B) The majority of PCNA molecules diffuses without constrains. 87% of the slowly diffusing (D < 0.1 μm2/s) and 54% of the fast diffusing (D > 0.1 μm2/s) mEos2-PCNA molecules reveal an unconfined motion. Trajectories of mEos2 monomers fused to a nuclear localization sequence (NLS, 30 kDa) reveal predominantly free diffusion (96%).

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References

    1. Chagin V. O., Stear J. H. & Cardoso M. C. Organization of DNA replication. Cold Spring Harbor Perspectives in Biology 2, a000737 (2010). - PMC - PubMed
    1. Moldovan G.-L., Pfander B. & Jentsch S. PCNA, the maestro of the replication fork. Cell 129, 665–679, 10.1016/j.cell.2007.05.003 (2007). - DOI - PubMed
    1. Majka J. & Burgers P. M. J. The PCNA-RFC families of DNA clamps and clamp loaders. Prog. Nucleic Acid Res. Mol. Biol. 78, 227–260, 10.1016/s0079-6603(04)78006-x (2004). - DOI - PubMed
    1. Naryzhny S. N. & Lee H. Characterization of proliferating cell nuclear antigen (PCNA) isoforms in normal and cancer cells: there is no cancer-associated form of PCNA. FEBS Lett. 581, 4917–4920, 10.1016/j.febslet.2007.09.022 (2007). - DOI - PubMed
    1. Casas-Delucchi C. S. & Cardoso M. C. Epigenetic control of DNA replication dynamics in mammals. Nucleus (Austin, Tex.) 2, 370–382, 10.4161/nucl.2.5.17861. - DOI - PubMed

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