Time-course analysis of Myc-RanGAP1 redistribution during LMB treatment.

<p>(A) HeLa cells were transfected with the plasmids encoding Myc-tagged RanGAP1 for 24 h, treated with 20 nM LMB for the indicated times, and analyzed by immunofluorescence microscopy using anti-Myc (9E10) and anti-RanBP2 antibodies. Bar, 10 μm. (B) The histogram indicates the percentages of cells with the indicated N/C ratios of Myc-RanGAP1 during the time-course analysis from three independent experiments. Each bar represents the mean value ± SEM (<i>N</i> = 50, Student’s <i>t</i> test). (C) The histogram indicates the percentages of cells with (+) or without (-) NPC staining of Myc-RanGAP1 at each indicated time point (<i>N</i> = 50, Student’s <i>t</i> test). For each experiment, 50 cells at each time point were used to analyze N/C ratio and NPC staining of Myc-RanGAP1.</p

The putative NLS sequence at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB.

<p>(A) The diagram shows mouse RanGAP1 wild-type (WT) (1–589) and its two C-terminal deletion mutants, CΔ23 (1–566) and CΔ49 (1–540). The mouse RanGAP1 consists of a leucine-rich repeat domain (LRR), an acidic region, and a SUMO-attachment domain (SUMO-AD) that contains a putative nuclear localization signal (NLS) (541–589) and also the lysine residue (K526) responsible for RanGAP1 SUMOylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141309#pone.0141309.ref022" target="_blank">22</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141309#pone.0141309.ref024" target="_blank">24</a>]. (B) The alignment of RanGAP homologs, including mouse RanGAP1 (P46061.2), human RanGAP1 (P46060.1), <i>Xenopus</i> RanGAP1 (O13066.1), <i>S</i>. <i>cerevisiae</i> Rna1p (P11745.2) and <i>S</i>. <i>pombe</i> Rna1p (P41391.1). (C) The NLS sequence of mouse RanGAP1 (541–589) is aligned with those of human RanGAP1 (539–587) and <i>Xenopus</i> RanGAP1 (532–580). (D-G) HeLa cells were transfected with the constructs encoding Myc-tagged RanGAP1 WT (D), CΔ23 (E), and CΔ49 (F), treated with 20 nM LMB or a control solution for 8 h, and analyzed by immunofluorescence microscopy with antibodies against RanBP2 and Myc or by immunoblotting with anti-Myc antibody (G). (H) The chart summarizes the characteristics of RanGAP1 WT, CΔ23, and CΔ49 in SUMOylation and localization in response to LMB treatment. C: Cytoplasm; N: Nucleoplasm; NPC: nuclear pore complex. Bar, 10 μm.</p

LMB-induced nuclear accumulation of Myc-RanGAP1 is reversible after removal of LMB.

<p>(A) HeLa cells transiently expressing Myc-tagged RanGAP1 were treated with 20 nM LMB for 8 h to induce a nuclear accumulation of RanGAP1. After removal of LMB, the cells were incubated with fresh medium for the indicated times and then analyzed by immunofluorescence microscopy using anti-RanBP2 and anti-Myc antibodies. Bar, 10 μm. (B) The histogram shows the percentages of cells with the indicated N/C ratios of Myc-RanGAP1 at each time point after removal of LMB. Each bar represents the mean value ± SEM (<i>N</i> = 50, Student’s <i>t</i> test). (C) The histogram indicates the percentages of cells with (+) or without (-) NPC staining of Myc-RanGAP1 at each time point after removal of LMB (<i>N</i> = 50, Student’s <i>t</i> test). The analyses were based on three independent experiments (B and C). For each experiment, 50 cells at each time point were used for analyzing both N/C ratio and NPC staining of Myc-RanGAP1.</p

Inactivation of CRM1 by LMB causes nuclear accumulation of RanGAP1 and a loss or decrease of RanGAP1 distribution in the cytoplasm and at the NPC.

<p>(A) HeLa cells were transfected with the plasmids encoding Myc-tagged RanGAP1 for 24 h, treated with 20 nM LMB or a control solution (0.3% DMSO in PBS) for 8 h and analyzed by immunofluorescence microscopy using mouse anti-Myc mAb (9E10) and rabbit anti-RanBP2 antibodies. (B) BRL cells were treated with 20 nM LMB or a control solution for 8 h and analyzed by immunofluorescence microscopy with mouse anti-RanGAP1 mAb (19C7). (C) BRL cells were treated with either 20 nM LMB for 8 h and 16 h or a control solution for 16 h followed by immunofluorescence microscopy with antibodies specific to RanBP2 and RanGAP1. Bar, 10 μm.</p

LMB treatment increases RanGAP1 SUMOylation in a RanBP2-independent manner.

<p>(A) HeLa cells were transfected with the plasmids encoding Myc-tagged RanGAP1 for 24 h, treated with 20 nM LMB or a control solution for 8 h, and analyzed by immunoblotting with mouse anti-Myc (9E10) and anti-α-tubulin mAbs. (B and C) BRL cells were treated with 20 nM LMB or a control solution for 8 h (B) and 16 h (C) followed by immunoblot analysis with mouse anti-RanGAP1 (19C7) and anti-α-tubulin mAbs. (D) HeLa cells were transfected with control or RanBP2-specific siRNA for 72 h, treated with 20 nM LMB or a control solution for 8 h, and then analyzed by immunoblotting with antibodies against RanGAP1, RanBP2 and α-tubulin.</p

RNAi-mediated depletion of CRM1 results in nuclear accumulation of RanGAP1 in mammalian cells.

<p>(A) HeLa cells were transfected with control siRNA or CRM1-specific siRNA 1 or 2 for 48 h followed by immunoblotting using antibodies specific CRM1 and α-tubulin. (B) HeLa cells were transfected with control siRNA or one of the two CRM1-specific siRNAs for 24 h and then the plasmids encoding Myc-tagged mouse RanGAP1 for 24 h followed by immunofluorescence microscopy analysis with mouse anti-Myc mAb (9E10). Bar, 10 μm. (C) The histogram shows the percentage of cells exhibiting the mean nuclear to cytoplasmic concentration ratio (N/C) of Myc-RanGAP1 in control or CRM1 RNAi cells. The immunofluorescence signal intensities of Myc-RanGAP1 in the nucleoplasm and cytoplasm were measured by ImageJ software (NIH) using cells from three independent experiments. Each bar represents the mean percentage value of cells with the indicated N/C ratio ± SEM for each treatment (<i>N</i> = 40, **<i>*P</i> < 0.001, Student’s <i>t</i> test).</p

A model shows how ALPCs affects nuclear import and export in the cytoplasm.

<p>ALPCs may serve as the docking or assembling sites for importin α/β-mediated import complexes followed by their dissociation for nuclear import. On the other hand, the ALPC-associated RanBP2/RanGAP1*SUMO1/Ubc9 complexes may function in the disassembly of CRM1-mediated export complexes by mediating RanGTP hydrolysis.</p

The ALPC-associated RanBP2/RanGAP1*SUMO1/Ubc9 complexes are distributed within the network of ER but not the tips of cell extensions.

<p>(A-D) HeLa cells were double stained with mAb414 and calreticulin antibody for labeling ER network (A), mAb414 and RanBP2 antibody (B), tubulin and calreticulin antibodies (C), and tubulin and RanBP2 antibodies (D) followed by immunofluorescence microscopy. The enlarged versions of inlets are shown at the bottom or top corner of each image (A-D). The arrows indicate the positions of the ALPC-associated RanBP2/RanGAP1*SUMO1/Ubc9 complexes that are most distant from the corresponding nucleus (D). The immunofluorescent images were taken using Olympus inverted IX81 widefield fluorescence microscope with U-Plan S-Apo 60×/1.35 NA oil immersion objective. Bar, 10 μm.</p

Ubc9 co-localizes with SUMO1-modified RanGAP1 and RanBP2 at both NPCs and ALPCs.

<p>(A) HeLa cells were analyzed by immunofluorescence microscopy using anti-Ubc9 antibody and mAb414. (B and C) HeLa cells were transfected with the construct encoding Myc-tagged Ubc9, double-stained with mouse anti-Myc mAb (9E10) and rabbit anti-RanGAP1 antibodies or with rabbit anti-Myc antibody and mouse anti-RanBP2 mAb, and then analyzed by immunofluorescence microscopy. Bar, 10 μm. The enlarged versions of inlets are shown at the top-right corner of each image.</p

CRM1-mediated export complexes accumulate at ALPCs when the disassembly of these export complexes is inhibited by transient expression of RanQ69L mutant.

<p>(A and B) HeLa cells were transfected with the construct encoding FLAG-tagged Ran wild-type (WT) or RanQ69L GTPase mutant (Mut), double-labeled with anti-FLAG and RanGAP1 antibodies (A) or mAb414 (B) and analyzed by immunofluorescence microscopy. (C-E) HeLa cells were co-transfected with the constructs encoding Rev-GR-GFP and FLAG-tagged Ran WT or RanQ69L. The transfected cells were incubated with 1 μM dexamethasone to induce the nuclear accumulation of Rev-GR-GFP, washed with fresh medium to remove dexamethasone to initiate nuclear export, stained with anti-RanGAP1 antibodies (C), anti-FLAG antibodies (D), or both anti-RanGAP1 and anti-FLAG antibodies (E), and analyzed by immunofluorescence microscopy. Bar, 10 μm. The boxes at the top-left corner of each image reveal the enlarged version of inlets.</p