Phosphorylation of <i>Xenopus</i> p31^comet potentiates mitotic checkpoint exit

<p>p31^comet plays an important role in spindle assembly checkpoint (SAC) silencing. However, how p31^comet's activity is regulated remains unclear. Here we show that the timing of M-phase exit in <i>Xenopus</i> egg extracts (XEEs) depends upon SAC activity, even under conditions that are permissive for spindle assembly. p31^comet antagonizes the SAC, promoting XEE progression into anaphase after spindles are fully formed. We further show that mitotic p31^comet phosphorylation by Inhibitor of nuclear factor κ-B kinase-β (IKK-β) enhances this role in SAC silencing. Together, our findings implicate IKK-β in the control of anaphase timing in XEE through p31^comet activation and SAC downregulation.</p

RCC1 regulates inner centromeric composition in a Ran-independent fashion

<p>RCC1 associates to chromatin dynamically within mitosis and catalyzes Ran-GTP production. Exogenous RCC1 disrupts kinetochore structure in <i>Xenopus</i> egg extracts (XEEs), but the molecular basis of this disruption remains unknown. We have investigated this question, utilizing replicated chromosomes that possess paired sister kinetochores. We find that exogenous RCC1 evicts a specific subset of inner KT proteins including Shugoshin-1 (Sgo1) and the chromosome passenger complex (CPC). We generated RCC1 mutants that separate its enzymatic activity and chromatin binding. Strikingly, Sgo1 and CPC eviction depended only on RCC1's chromatin affinity but not its capacity to produce Ran-GTP. RCC1 similarly released Sgo1 and CPC from synthetic kinetochores assembled on CENP-A nucleosome arrays. Together, our findings indicate RCC1 regulates kinetochores at the metaphase-anaphase transition through Ran-GTP-independent displacement of Sgo1 and CPC.</p

Upregulation of annulate lamellae by RNAi-knockdown of ELYS decreases the rates of both nuclear import and export.

<p>HeLa cells were transfected with control or ELYS-specific siRNAs and then with the construct encoding Rev-GR-GFP. (A and B) To evaluate the effect of ELYS RNAi on the rate of nuclear import, the transfected cells were treated with 0.25 μM of dexamethasone for the indicated times and analyzed by fluorescence microscopy (A). The histogram shows the nuclear to total signal ratios of Rev-GR-GFP (B). (C and D) To test if ELYS RNAi affects the rate of nuclear export, the transfected cells were treated with 0.25 μM of dexamethasone for 3 h to induce the nuclear accumulation of Rev-GR-GFP, washed with PBS, incubated with fresh medium for the indicated times, and analyzed by fluorescence microscopy (C). The histogram shows the cytoplasmic to total signal ratio of Rev-GR-GFP (D). Each bar indicates the mean value ± SEM (<i>N</i> = 60, Student’s <i>t</i> test) (B and D). Bar, 10 μm (A and C).</p

SUMO1-modified RanGAP1 is equally distributed between the nuclear and cytosolic fractions in a variety of mammalian cells.

<p>(A) HeLa cells were fractionated by two different methods using either digitonin or NP-40 as non-ionic detergent. The nuclear and cytosolic fractions were analyzed by immunoblotting with antibodies specific to SUMO-1, RanGAP1, α-tubulin as a marker for cytosolic proteins, lamin B as a marker for nuclear proteins, or POM121 as a marker for the NPC proteins. (B and C) Different types of tumor/cancer cells including HeLa, BRL, 293T and U2OS (B) and normal/non-tumorigenic fibroblasts including human GM03652 and mouse NIH3T3 (C) were fractionated by NP-40 method and then analyzed by immunoblotting with the indicated antibodies. (D) Total cell lysates of HeLa, PN and SMC cells were used for immunoblot analysis with the indicated antibodies.</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

ALPCs function as intermediate docking sites for importin α/β-mediated import complexes during nuclear import.

<p>HeLa cells were transfected with siRNAs specific to ELYS to upregulate annulate lamellae and then with the construct encoding Rev-GR-GFP fusion proteins. The transfected cells were incubated with LMB for 2 h to inhibit CRM1-mediated export, treated with both dexamethasone (1 μM) and LMB to induce importin α/β-mediated import for the indicated times, and analyzed by immunofluorescence microscopy. At least 50 Rev-GR-GFP cells were analyzed for each time point of dexamethasone treatment to select o representative cell as shown in this figure. The arrows indicated the sites of ALPCs. Bar, 10 μm.</p

SUMO1-modified RanGAP1 localizes to both NPCs and ALPCs in a variety of mammalian cells.

<p>(A) The diagram shows that compared to NPCs in the nuclear envelope, ALPCs are embedded in the membrane cisternae of annulate lamellae that are often connected to the membrane network of ER. (B) Human cervical cancer cells (HeLa) were double-labeled with anti-RanGAP1 antibody and anti-SUMO1 mAb (21C7) or mAb414 for staining NPCs and ALPCs and then analyzed by immunofluorescence microscopy. (C) Mouse embryonic fibroblasts (NIH3T3) were double-stained with anti-RanGAP1 antibody and mAb414 or anti-RanBP2 mAb. (D) Rat primary cortical/hippocampal neurons (PN) were double-labeled with anti-RanGAP1 antibody and mAb414. (E) Human bronchial/tracheal smooth muscle cells (SMC) cells were double-stained with anti-RanGAP1 antibody and mAb414 or anti-RanBP2 mAb. Bar, 10 μm. The boxes at the top corner of each image show an enlarged version of inlets. (F) Annulate lamellae are highly abundant in SMC cells. 60 SMC cells were double-stained with anti-RanGAP1 antibody and mAb414. All the ALPC foci in each cell were counted under Olympus inverted IX81 fluorescence microscope using Z-stacks. The number of ALPC foci per cell was classified into three categories (10–50, 50–100 and ≥100), and the percentage of cells in each category was indicated. Each column represents the mean value ± SEM (<i>N</i> = 60) (ALPC foci/cell: 10–182; Average = 63).</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

Both covalent SUMOylation and non-covalent interaction with RanBP2 are required for RanGAP1 localization to ALPCs.

<p>(A) HeLa cells were transfected with the constructs encoding Myc-tagged RanGAP1 wild-type (WT) or SUMOylation-deficient K526R mutant (Mut) and analyzed by immunofluorescence microscopy with antibodies specific to Myc and RanBP2. The boxes at the bottom corner of each image show the enlarged version of inlets. Bar, 10 μm. (B) The transfected cells were analyzed by immunoblotting with antibodies specific to RanGAP1, Myc and α-tubulin. (C) HeLa cells were transfected with control or RanBP2-specific siRNAs, double-stained with antibodies specific to RanBP2 and RanGAP1, and analyzed by immunofluorescence microscopy. In the lower panel, white dashed lines indicate the borders of RanBP2 RNAi cells, in which “-” indicates a significant knockdown of RanBP2 and “+” indicates that the signals of RanBP2 are comparable to those in control RNAi cells (upper panel). Bar, 10 μm. (D) The cells transfected with control or RanBP2-specific siRNAs were analyzed by immunoblotting with the indicated antibodies.</p