Model of ORC dynamics. (A) ORC may exist as a stable complex that dynamically associates with chromatin. (B) Alternatively, ORC may exist as a more stable chromatin-associated complex, consisting of dynamic subunits. Details are contained within the text.

GFP-ORC1 co-fractionates with endogenous ORC1 and chromatin. (A) Immunoblots of whole cell extracts prepared from a GFP-ORC1 expressing cell line (3KA4) grown either in the presence or absence of tetracycline were probed with anti-CgORC1 antibodies. (B) Nuclei isolated from 3KA4 cells grown in the absence of tetracycline were incubated with different concentrations of salt (150 mM v 300 mM), and soluble and insoluble fractions were probed with anti-CgORC1. Additional characterization of this cell line can be found in Okuno et al. [3]. (C) Isolated chromatin from 3KA4 was digested with micrococcal nuclease and the soluble fraction concentrated with a 300 kDa molecular weight cut-off ultrafiltration device. GFP-ORC1, endogenous ORC1 and ORC2 were all present in the greater than 300 kDa fraction. Shown are the flow-through (FT) and high molecular weight fraction (HMWF). (D) GFP-ORC1 co-fractionates with endogenous ORC1 and chromatin throughout the cell-cycle. Induced 3KA4 cells were synchronized and fractionated as described in Fig. 1 and immunoblots probed with anti-CgORC1.

FLIP of GFP-CgORC1 and GFP-HP1α, corrected for nuclear volume. (A) FLIP analysis of GFP-CgORC1 during the cell-cycle. GFP-CgORC1 expressing cells were subjected to a repeating cycle of an image scan, a 0.5 s bleach pulse at 100% laser power and a 5 s recovery period. The images are from a typical FLIP experiment showing the loss of GFP fluorescence over time as a result of the repeated bleach pulse (indicated by the cross). (B) FLIP kinetic plot of GFP-CgORC1 fluorescence loss as a function of time. Each line represents the average fluorescence loss from at least 5 cells, with the loss quantified at 2–3 distinct sites within the nucleus of each cell, at the indicated times during the cell-cycle. The error bars indicate the standard deviation among the individual measurements for each timepoint. Similar results were obtained in three independent experiments. Results with EGFP and histone H1c are provided as reference marks for in vivo protein mobility. (C) FLIP analysis of a stable CHO cell line expressing GFP-HP1α, performed as in panel (A). (D) FLIP kinetic plot for GFP-HP1α during the cell-cycle. Data are displayed as in panel (B). Similar results were obtained in three independent experiments.

The effect of nuclear volume on Fluorescence loss in photobleaching kinetics. (A) Initial FLIP kinetic plot of the GFP-ORC1 expressing cell line, 3KA4 demonstrating fluorescence loss as a function of time. 3KA4 cells were synchronized and released at the indicated timepoints as described in Materials and methods. Cells were subjected to a repeating cycle of an image scan, a 0.5 s bleach pulse at 100% laser power and a 5 s recovery period. Each line represents the average fluorescence loss from at least 5 cells, with the loss quantified at 2–3 distinct sites within the nucleus of each cell, at the indicated time during the cell-cycle. Similar results were obtained in three independent experiments. Results with GFP-tagged histone H1c are provided as a reference mark for in vivo protein mobility. (B) FLIP analysis of a stable cell line expressing free-EGFP was performed as described in panel (B). (C) The relative size of the nucleus expands as cells progress through the cell-cycle. The area of the nucleus at its largest diameter was measured at each timepoint from multiple cell lines and averaged. (D) Schematic representation of the effect of increasing nuclei size on photobleaching kinetics. Details are contained within the text.

Cell line characterization and FLIP of ORC4. (A) Homogeneity and inducibility of GFP-P-ORC4. PC1–11 cells (CHO cells stably expressing GFP-P-ORC4) were grown either in the presence (uninduced—gray) or absence (induced—orange) of tetracycline and analyzed for EGFP expression by FACS. Untransfected CHO cells (purple) were used as a negative control. (B) The expression of GFP-P-ORC4 is inducible (left panel) and GFP-P-ORC4 exhibits similar salt extractability as endogenous ORC4 (right panel). Whole cell extracts from induced and uninduced PC1-11 cells were run on 10% SDS-PAGE gels and immunoblotted with anti-ORC4 demonstrating induction of full-length GFP-P-ORC4 (left panel). PC1-11 cells were induced and isolated nuclei fractionated using increasing levels of NaCl (150 mM and 300 mM) and the resulting fractions were probed with anti-ORC4 antibodies (right panel). (C) Typical images from the cell-cycle FLIP of GFP-P-ORC4 in PC1-11 as in Figs. 4A and C. (D) FLIP kinetic plot from the cell-cycle FLIP analysis of GFP-P-ORC4, as described in Fig. 4. The error bars represent the standard deviation from one experiment. For each experiment, at least 5 cells were photobleached with 2–3 measurements taken per cell. EGFP and histone H1c are provided as reference marks for mobility. Similar results were obtained in three independent experiments. (E) Comparative summary of the relative FLIP kinetics for GFP-ORC1 (ORC1), GFP-P-ORC4 (P-ORC4), ORC4 (without the peptide linker) and HsHP1α.

ORC is stable and chromatin-associated throughout the cell-cycle in CHO cells. (A) CHO cells were synchronized in mitosis with a brief (4 h) nocodazole block and collected and fractionated at the indicated timepoints as described in Materials and methods. Cell extracts were subjected to electrophoresis on 8% SDS-PAGE gels and immunoblotted with anti-ORC1, 2 and 4. Anti-geminin and anti-actin were used as controls for mitotic release and protein levels. The S2 and S3 fractions represent soluble protein fractions and the P3 fraction represents the chromatin-enriched fraction. (B) CHO cells were synchronized in S-phase and cells fractionated at 0, 1, 4, 8 or 11 h as described in Materials and methods. As a control for S-phase progression, extracts were immunoblotted with anti-cyclin B1. Anti-actin was used as a loading control. Similar results were obtained in 4 independent experiments (3 times with aphidicolin and once with mimosine arrested cells). The results of two of these experiments were additionally confirmed using an anti-HsORC1 antibody (gift of J. Mendez and B. Stillman, not shown). (C) To monitor S-phase progression, aliquots of cells from (B) were pulse labeled with BrdU for 30 min, fixed and stained with anti-BrdU. The percentage of cells displaying the indicated replication pattern (^ Early, ▪ Middle, ▴ Late) was scored. (D) CHO cells synchronized in S-phase as in panel (B) were released into media containing: DMSO (control), 50 μg/ml cycloheximide or 25 μM MG132 for 0, 1, 4, 8 and 11 h. Whole cell extracts were prepared and immunoblotted for ORC1, 2, 4, SP1 and actin. Similar results were obtained in two independent experiments.