The dynamic association of RCC1 with chromatin is modulated by Ran-dependent nuclear transport

Abstract

Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or alpha-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo.

Figure 1.

Comparison of GFP-RCC1 with endogenous RCC1. (A) Anti-RCC1 immunoblot showing the expression levels of RCC1 and GFP-RCC1. Left lane, tsBN2 cells grown at permissive temperature (33°C). Middle lane, tsBN2 cells after being shifted to nonpermissive temperature (40°C) for 15 h. Right lane, GFP-RCC1 tsBN2 cells grown at 40°C. The same amount of cells is loaded on each lane. (B) GFP-RCC1 colocalizes with chromatin throughout the cells cycle. Left panel, GFP-RCC1 in a living cell. Middle panel, DNA stained with Hoescht stain in the same cell. Right panel, overlay of these two images. The top row shows an interphase cell, whereas the bottom row shows a cell in metaphase (C) RCC1 and GFP-RCC1 require the same salt concentration for elution from chromatin. Top gel, extraction of RCC1 from tsBN2 cells (grown at 33°C) with increasing concentrations of NaCl. Bottom gel, GFP-RCC1 is extracted from GFP-RCC1 cells (grown at 40°C) the same concentration of NaCl. After salt incubation, the samples were separated into supernatants (S) and pellets (P) and both were blotted with an anti-RCC1 antibody.

Figure 4.

Excess Ran slows GFP-RCC1 recovery after photobleaching. (A) GFP-RCC1 cells microinjected with injection buffer (top) or the mutant T24N Ran (bottom) before photobleaching and measurement of fluorescence recovery. The red fluorescence is the Texas Red 70-kDa dextran coinjected with buffer or unlabeled Ran in the cytoplasm to mark the injection site. The green fluorescence is GFP-RCC1. (B) Recovery of fluorescence after photobleaching for GFP-RCC1 interphase cells either uninjected (blue diamonds), microinjected with marker alone in injection buffer (pink squares), or marker plus wt Ran (yellow triangles), Q69L Ran (green Xs), or T24N Ran (purple asterisks). After photobleaching, the fluorescence was measured every second for 120 s. (C) The t_1/2 of recovery for each condition calculated from the data points shown in B. n = 15 for each condition and the SEs are shown (SEM). The times of recovery of interphase cells injected with wt Ran, Q69L Ran, and T24N Ran were significantly different from cells injected with buffer alone (p<.05). The recovery time for T24N Ran was significantly different from that for wt Ran (p<.001), however the recovery time for Q69L Ran was not significantly different from wt Ran (p=0.076). In these experiments 5 microinjected cells from 3 separate coverslips (total of 15 cells for each condition) were blindly selected for FRAP analysis. The analyzed recovery period was extended to 2 min (compared with 1 min in the experiments shown in Figure 2) to allow complete recovery of all cells.

Figure 2.

GFP-RCC1 is highly mobile. A rectangular region of GFP-RCC1 cells was photobleached as described in the MATERIALS AND METHODS. The fluorescence inside the photobleached rectangle was then measured every 1 s for 60 s to determine the rate of recovery. (A) A comparison of the recovery rate of GFP-RCC1 fluorescence after photobleaching in interphase nuclei (top and bottom row) and a cell in metaphase (middle row). The cell in the bottom row was first energy depleted by incubation with sodium azide and 2-deoxyglucose in gluc (-) media for 20 min before observation. (B) Quantitation of the recoveries after photobleaching of GFP-RCC1 in interphase cells, metaphase cells, and energy-depleted interphase cells. Each point represents the average of 15 cells. (C) Average t_1/2 (amount of time required after photobleaching to return to 50% of the final fluorescent intensity) of interphase, metaphase, and energy-depleted cells calculated from the data shown in B. The GFP-RCC1 recovery times for metaphase and energy-depleted cells were significantly different from interphase cells, p < 0.001, n = 15).

Figure 3.

GFP-RCC1 becomes highly clumped inside the nucleus upon energy depletion. Cells expressing GFP-RCC1 and growing in normal media were assembled in a Bioptechs live-cell chamber with normal media and allowed to equilibrate in a 37°C incubator. After 20 min, the chamber was transferred to a DeltaVision microscopy work station and images were acquired. The media were changed to gluc (-) media, and the same cell was imaged again after 20 min. Finally, the media were changed to gluc (-) media containing sodium azide and deoxyglucose and the same cell was imaged after 20 min in this ATP depletion media. To show the DNA staining in live, unfixed cells, cells were incubated in 1 μg/μl Hoechst 33258 for 20 min before image acquisition. Shown in the figure are single, deconvolved z-sections.

Figure 5.

Agents that disrupt various Ran-dependent export pathways also alter the mobility of GFP-RCC1. GFP-RCC1 tsBN2 cells were pretreated for 5 h (at 40°C) with the indicated agent before photobleaching. (A) Measurement of the t_1/2 required for recovery of fluorescence after photobleaching after the various treatments. Cells were incubated for 5 h at 40°C before FRAP analysis with either LMB (50 ng/ml), low actinomycin D (0.02 μg/ml), high actinomycin D (3.2 μg/ml), low α-amanitin (50 μg/ml), or high α-amanitin (300 μg/ml). (B) The extent of recovery after photobleaching after no treatment (purple diamonds), LMB (red squares), high α-amanitin (orange triangles), high actinomycin D (green squares), low actinomycin D (yellow diamonds), and low α-amanitin (blue circles). The cells treated with high actinomycin D did not recover to the same level as the others, indicating the likely presence of a second, more immobile population of GFP-RCC1. In Figures 2B and 4B, 1.0 on the y-axis of the recovery graphs represents the final recovery achieved after photobleaching. In Figure 5B, 1.0 on the y-axis represents the initial fluorescence before photobleaching.

Figure 6.

Immunofluorescence microscopy showing the effects of these various treatments on the localization of four cellular proteins. GFP-RCC1 tsBN2 cells were either energy depleted with Na azide and 2-deoxyglucose in gluc (-) media for 30 min at 40°C, or incubated at 40°C for 5 h with either LMB (50 ng/ml), low actinomycin D (0.02 μg/ml), high actinomycin D (3.2 μg/ml), low α-amanitin (50 μg/ml), or high α-amanitin (300 μg/ml). After treatment, the cells were fixed and processed for indirect immunofluorescence microscopy by using primary antibodies to Ran, the nuclear export carrier Crm1, the mRNA splicing factor SRm160, or the nucleolar protein B23/nucleophosmin followed by a TRITC- or Texas Redlabeled second antibody. Distribution patterns that differ from the pattern in untreated cells are marked with stars.

Figure 7.

Efficient GFP-RCC1 release from chromatin in permeabilized GFP-RCC1 tsBN2 cells requires cytosol and energy. (A) After permeabilization with 0.1% Triton X-100 and rinsing in buffer A, cells were either fixed immediately (top row) or incubated with buffer A containing 0.4 M NaCl for 20 min on ice before rinsing and fixation. GFP-RCC1 was localized with an anti-GFP first antibody and TRITC-labeled second antibody (left) and the cells were counterstained with 4,6-diamidino-2-phenylindole to localize the DNA. (B) After permeabilization, cells were incubated for 20 min at RT with the indicated additions before washing, fixation, and indirect immunofluorescence. GTP and Xenopus ovarian cytosol (cyt) were added at 1 mM and 11.5 mg/ml, respectively. wt, Q69L, or T24N Ran was added at 10 μM. Detection and quantitation of GFP-RCC1 was as described in MATERIALS AND METHODS. Shown for each condition are the mean and SEM of the nuclear fluorescence of between 30 and 90 nuclei. All values are expressed relative to the 100% (fixed after permeabilization) and the 0% (fixed after stripping with 0.4 M NaCl) controls. Note that the inclusion of 10 μM Ran decreased the loss obtained with cytosol alone (asterisks). (C) A concentration curve measuring the release of GFP-RCC1 obtained with increasing amounts of cytosol either in the presence (•) or absence (○) of 1 μM Ran. All samples contained 1 mM GTP.

Figure 8.

The nucleotide requirements for GFP-RCC1 release from chromatin in permeabilized cells. Permeabilized GFP-RCC1 cells were incubated for 20 min at RT with the indicated addition before washing and fixation. Each indicated nucleotide was added at 1 mM. Cytosol (cyt) was added at 11.5 mg/ml. Detection and quantitation of GFP-RCC1 was as described in the MATERIALS AND METHODS. Between 40 and 90 nuclei were quantitated for each condition to yield the mean and SEM.