Li HY et al. (2003), A mechanism of coupling RCC1 mobility to RanGTP...

XB-ART-5706

J Cell Biol 2003 Mar 03;1605:635-44. doi: 10.1083/jcb.200211004.

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A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo.

Li HY , Wirtz D , Zheng Y .

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The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1-Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface.

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Species referenced: Xenopus laevis
Genes referenced: h2bc21 mtor ran rcc1

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	Figure 1. Characterization of RCC1-GFP. (A) RCC1-GFPâexpressing or ânonexpressing Swiss 3T3 cells were fixed and stained with DAPI to visualize DNA. The nonexpressing cells were also stained with anti-RCC1 antibody to visualize endogenous RCC1. Both RCC1-GFP and endogenous RCC1 colocalized with DAPI in interphase and mitosis. Bar, 10 Î¼m. (B) Time-lapse microscopy of RCC1-GFPâexpressing cells during mitosis. Bar, 10 Î¼m. (C) RCC1-GFP and endogenous RCC1 were extracted from isolated nuclei with increasing concentrations of salt and analyzed by Western blotting. (D) Plasmids expressing RCC1-GFP or GFP were transfected into tsBN2 cells, which harbor a temperature-sensitive mutation in RCC1, and the wild-type parental cells, BHK-21. The cells expressing RCC1-GFP or GFP were counted in five random fields with a 20Ã objective 24 h after transfection. Cells were then shifted to the nonpermissive temperature (39.5Â°C) to inactivate endogenous RCC1 in tsBN2 cells and were counted every 24 h. The tsBN2 cells transfected with GFP did not survive at the nonpermissive temperature. The same cells transfected with RCC1-GFP, and the control BHK-21 cells survived, leading to establishment of stable cell lines able to grow at 39.5Â°C.
	Figure 2. FRAP. (A) Selected images of an interphase cell during FRAP of an area in the nucleus (red circle). Bottom panels show the enlargement of the indicated area (white dashed square) in pseudocolor. (B) Selected images of a mitotic cell during FRAP of an area on the chromosome. Bar, 10 Î¼m. (C) RCC1-GFP FRAP kinetics in the interphase nucleus, on condensed chromosomes, and in the nuclei of methanol-fixed cells. (D) GFP and histone H2B-GFP FRAP kinetics in the interphase nucleus. Values in C and D represent means Â± SD from at least five different cells.
	Figure 3. FLIP. (A) Selected images of interphase or mitotic cells during FLIP of the indicated area (yellow circles). Bars, 10 Î¼m. (B) Kinetics of overall RCC1-GFP FLIP in interphase and mitotic cells. (C and D) Kinetics of RCC1-GFP FLIP in interphase (C) and mitotic (D) cells.
	Figure 4. The binary complex of RanâRCC1 binds stably to the chromatin in vivo. (A) Rh-RanGDP, Rh-RanT24N, or Rh-RanGTP was injected into interphase nuclei or mitotic cytoplasm of RCC1-GFPâexpressing cells, followed by live imaging using fluorescence microscopy. Bar, 10 Î¼m. (B) The model. RCC1, RanGDP, and RanGTP interact with the chromatin reversibly due to low affinity binding, whereas the RCC1âRan binary complex binds to the chromatin stably. The ternary complexes of RCC1âRanâGDP (or GTP) are omitted from the drawing for simplicity. (C) RanGDP or RanT24N was injected into the interphase nuclei or mitotic cytosol at 1 mg/ml followed by FRAP analysis of RCC1-GFP. Selected images before and after the bleach pulses (red circle denotes the bleached spot) were shown. Colored panels show the enlargement of the indicated area (white dashed square) in pseudocolor. Bar, 10 Î¼m. (D) FRAP kinetics of injected interphase cells. Values represent means Â± SD from at least five cells. (E) FRAP kinetics of injected mitotic cells. Values represent means Â± SD from at least three cells and five independent FRAPs.
	Figure 5. Biochemical characterization of the binding of Ran and RCC1 to the chromosomes. (A) Both RanGDP and RanT24N strongly stimulate the binding of the endogenous RCC1 in the egg extract to the mitotic chromosomes assembled from the sperm chromatin. The binding of the endogenous RCC1 to the chromatin is detected by immunofluorescence using an anti-RCC1 antibody. (B) RCC1-GFP and sperm was added to the egg extract supplemented with purified RanGDP or RanT24N. The fluorescence intensity of RCC1-GFP on the sperm chromatin was quantified as arbitrary unit (AU). (C) RCC1 competition. The sperm was incubated with egg extracts supplemented with RCC1-GFP and either RanGDP or RanT24N in the presence of unlabeled RCC1 at the indicated concentrations. The amount of RCC1-GFP bound to the chromatin was quantified. (D) Ran competition. Sperm was incubated with egg extracts containing Rh-RanGDP or Rh-RanT24N and unlabeled RanGDP or RanT24N at the indicated concentrations, respectively. The amount of labeled Ran bound to the chromatin was quantified. (E and F) Buffer containing 0â5 mM GTP was incubated with sperm chromatin with bound Rh-Ran or RCC1-GFP on coverslips for 20 min. The amount of labeled proteins on the sperm chromatin was quantified either before (c, control) or after incubation. (G and H) The same sperm chromatin as in E and F were incubated with buffer containing indicated concentrations of unlabeled Ran and RCC1 as competitors in the absence of free GTP. The amount of chromatin-bound RCC1-GFP (G) or Rh-Ran (H) was quantified after incubation. Error bars represent SD.

References [+] :

Bilbao-Cortés, Ran binds to chromatin by two distinct mechanisms. 2002, Pubmed, Xenbase