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Many mitotic factors were shown to be activated by Ran guanosine triphosphatase. Previous studies in Xenopus laevis egg extracts and in highly proliferative cells showed that mitotic chromosomes were surrounded by steep Ran guanosine triphosphate (GTP) concentration gradients, indicating that RanGTP-activated factors promote spindle assembly around chromosomes. However, the mitotic role of Ran in normal differentiated cells is not known. In this paper, we show that although the steep mitotic RanGTP gradients were present in rapidly growing cell lines and were required for chromosome congression in mitotic HeLa cells, the gradients were strongly reduced in slow-growing primary cells, such as HFF-1 fibroblasts. The overexpression of RCC1, the guanine nucleotide exchange factor for Ran, induced steeper mitotic RanGTP gradients in HFF-1 cells, showing the critical role of RCC1 levels in the regulation of mitosis by Ran. Remarkably, in vitro fusion of HFF-1 cells produced cells with steep mitotic RanGTP gradients comparable to HeLa cells, indicating that chromosomal gain can promote mitosis in aneuploid cancer cells via Ran.
Figure 2. Differences in RCC1 levels and N-terminal methylation contribute to the cell-specific diversity of mitotic RanGTP gradients. (A and B) Immunoblots showing protein expression levels and posttranslational modifications in nonsynchronized cells (A) and in HFF-1 and HeLa cells synchronized by NZ shake-off in mitosis or interphase (B). As indicated, the position of methylated RCC1 (Me3-RCC1α) and phosphorylated RanGAP1 (p-RanGAP1-SUMO) are shown. Corresponding loading control data for each separate gel are shown. Asyn, asynchronized; Inter, interphase; Mito, mitosis. (C) IF of RCC1 in HeLa and HFF-1 cells. (D) Scatter plot of RCC1 chromatin/cytoplasmic ratio detected by IF in mitotic HeLa and HFF-1 cells. (E) Confocal fluorescence images of live cells expressing NTDα-mCherry. (F) Scatter plot of chromatin/cytoplasmic ratio of NTDα-mCherry in live cells. (G) FRAP measurements of wt and ASPK RCC1α-mCherry binding to chromatin in interphase (left) and mitotic cells (right). D, F, and G are single-cell data, means ± SD; t test in D; ANOVA/Bonferroni in F and G. In F, all pairwise comparisons are significant (P < 0.01), except for two pairs indicated (NS). In G, only significant pairwise comparisons are indicated. (H) mCherry fluorescence, donor intensity (Idonor), and FLIM images of mitotic HFF-1 cells coexpressing wt (top) or S2K RCC1α-mCherry and Rango-4. Bars, 10 µm.
Figure 3. Mitotic RanGTP gradient promotes rapid spindle assembly during PM. (A and B) Time-lapse imaging of untreated cells was used to measure the duration of mitotic phases (see also Fig. S3 A). (A) Scatter plot of PM/total mitosis time in HeLa and HFF-1 cells (single-cell data, means ± SD, t test). (B) Linear regression of the PM versus metaphase time in HeLa and HFF-1 cells. Best-fit regression lines and the corresponding p-values of the fits are indicated. (C) RCC1 IF in control or NRMT RNAi-treated HeLa cells. (D) Rango-4 donor intensity (Idonor) and FLIM images of control or NRMT RNAi-treated HeLa cells. (E) Scatter plot of mitotic cargo gradients and mean cellular Rango-4 E. Single-cell data, means ± SD, t test. (F and G) The fraction of mitotic cells in PM, metaphase, anaphase, and telophase (A-T) detected by IF in NRMT or control RNAi-treated HeLa cells (F) and in HFF-1 cells expressing or not expressing S2K RCC1α-mCherry (G). CD, chromosome congression defect; LC, lagging chromosomes; 2P, two poles or asters; >2P, multipolar spindles and asters. Means ± SD, ANOVA/Bonferroni test; n = 4 in F and n = 3 in G. Only significant changes are indicated. Bars, 10 µm.
Figure 4. Chromosomal gain drives steep mitotic RanGTP gradient. (A) mCherry fluorescence, donor intensity (Idonor), and FLIM images of mitotic cells resulting from the fusion of HFF-1 cells expressing EB3-mCherry with HFF-1 cells expressing RBP-4 (bottom). In the control HFF-1 cells expressing RBP-4 (top), the PEG-induced cell fusion was omitted. The displayed Ïdonor range is shown on the right. (B) Scatter plot of the mitotic RanGTP gradients (RBP-4 ÎE) in control and fused HFF-1 cells (single-cell data, means ± SD, t test). Bar, 10 µm.
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