Cell cycle-dependent binding modes of the ran exchange factor RCC1 to chromatin

Abstract

The formation of an activity gradient of the small G-protein Ran around chromatin depends on the differential partitioning of the opposing enzyme activities of the Ran guanine nucleotide exchange factor RCC1 that resides on chromatin, and the cytoplasmic Ran GTPase activating protein RanGAP. We studied the time-dependent interaction kinetics between RCC1 and chromatin and the mobility of the Ran-RCC1 complex in living cells by fluorescence correlation spectroscopy to investigate whether binding of RCC1 to chromatin regulates the exchange activity of RCC1, and whether the stability of the RCC1-chromatin interaction is regulated during the cell cycle. We found that RCC1 mobility is dominated by two states: a highly mobile state that is trapped within chromatin, and a transiently immobilized state that is stabilized during mitosis. We show that only the immobilized state of RCC1 interacts with Ran and conclude that its guanine nucleotide exchange activity is restricted to specific sites on chromatin.

Figure 1

Autocorrelation curves of RCC1-EGFP on chromatin and in the cytoplasm of HeLa cells at different stages in the cell cycle. Representative measured in the nucleus of an interphase HeLa cell (A), the cytoplasm of an interphase HeLa cell (B), on chromatin in a HeLa cell in metaphase (C), and in the cytoplasm of a HeLa cell in metaphase (D). (Curves in panels A and C were fit with the binding-diffusion model; curves in panels B and D were fit with a model for free diffusion.) (Upper panel of each graph) Fit residuals. (Insets) Confocal images of RCC1-EGFP expressing HeLa cells, with measurement positions indicated (open crosses). Scale bars: 5 μm.

Figure 2

Diffusion constant and off-rate constant of RCC1-EGFP as function of temperature measured in nuclei of HeLa cells. (Top panel) Diffusion constant of RCC1-EGFP (solid) and EGFP (shaded) as a function of temperature. (Center panel) Dissociation rate constant of RCC1-EGFP as a function of temperature. (Bottom panel) Free fraction as a function of temperature. Error bars indicate the SD from measurements in at least four cells.

Figure 3

Equilibration of RCC1 between chromatin and cytoplasm in mitotic cells. (A and B) A HeLa cell expressing RCC1-EGFP (green) and Tubulin-mCherry (red) was arrested in metaphase with 100 ng/mL nocodazole and fluorescence was imaged over a period of over 1 h. Scale bar: 5 μm. Average cytoplasmic and chromatin fluorescence intensities of RCC1-EGFP were used to calculate the fraction of unbound RCC1 at each time point (B). RCC1-paGFP was photoactivated on mitotic chromatin and the cytoplasmic fluorescence intensity of photoactivated RCC1-paGFP followed in time (C).

Figure 4

Determination of the fraction of unbound RCC1 in mitotic cells by fluorescence imaging. (A) Mitotic cells expressing different constructs of RCC1-EGFP were imaged using confocal fluorescence microscopy. (B) Fraction of unbound protein was calculated from average fluorescence intensity in cytoplasm and on chromatin. Error bars indicate SD from measurements in n cells. Scale bars: 5 μm.

Figure 5

Autocorrelation curves of EGFP-Ran and EGFP-RanT24N in the nucleus and in the cytoplasm of HeLa cells during interphase. (A) Autocorrelation curves of EGFP-Ran recorded in the cytoplasm (left) or the nucleus (right) of HeLa cells during interphase were fit with either a model for free diffusion (blue) or the binding diffusion model (red). (Upper panels) Fit residuals for each model. (B) Autocorrelation curve of EGFP-RanT24N recorded in the nucleus of an interphase HeLa cell and fit with the binding diffusion model. (Upper panel) Fit residuals. (C) Confocal images of HeLa cells expressing either EGFP-Ran (left column) or EGFP-RanT24N (right column) during interphase (top row) or mitosis (bottom row). Cell borders are outlined (thin white line). Scale bars: 5 μm.

Figure 6

Normalized interaction strengths for RCC1-Ran and RCC1-RanT24N complexes as determined by dual-color fluorescence cross-correlation spectroscopy. Measurements were taken either in interphase nuclei (I) or on mitotic chromatin (M). (Each box-and-whisker chart represents measurements from N different cells.)

Figure 7

Cross-correlation curves of RCC1-EGFP and mCherry-RanT24N on chromatin. Representative cross-correlation curves recorded in interphase nuclei (A) or on mitotic chromatin (B) of HeLa cells coexpressing RCC1-EGFP and mCherry-RanT24N were fit with the binding diffusion model (red curves) to determine the diffusion and dissociation rate of the RanT24N-RCC1 complex.