Fig 1.
SUMO1-modified RanGAP1 localizes to both NPCs and ALPCs in a variety of mammalian cells.
(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 (N = 60) (ALPC foci/cell: 10–182; Average = 63).
Fig 2.
Ubc9 co-localizes with SUMO1-modified RanGAP1 and RanBP2 at both NPCs and ALPCs.
(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.
Fig 3.
Both covalent SUMOylation and non-covalent interaction with RanBP2 are required for RanGAP1 localization to ALPCs.
(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.
Fig 4.
The ALPC-associated RanBP2/RanGAP1*SUMO1/Ubc9 complexes are distributed within the network of ER but not the tips of cell extensions.
(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.
Fig 5.
SUMO1-modified RanGAP1 is equally distributed between the nuclear and cytosolic fractions in a variety of mammalian cells.
(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.
Fig 6.
Upregulation of annulate lamellae causes a redistribution of both pore complexes and nuclear transport receptors from the nuclear envelope to annulate lamellae.
(A) HeLa cells were treated with vinblastine or DMSO as a control and analyzed by immunofluorescence microscopy with mAb414 and anti-RanGAP1 antibodies. (B-F) HeLa cells were transfected with control or ELYS-specific siRNAs, double-labeled with mAb414 and anti-ELYS antibodies (B), mAb414 and anti-RanGAP1 antibodies (C), anti-importin α and anti-RanGAP1 antibodies (D), anti-importin β and anti-RanGAP1 antibodies (E), and anti-CRM1 and anti-RanGAP1 antibodies (F) followed by immunofluorescence microscopy. The boxes at the corner of each image represent the enlarged version of inlets. Bar, 10 μm.
Fig 7.
Upregulation of annulate lamellae by RNAi-knockdown of ELYS decreases the rates of both nuclear import and export.
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 (N = 60, Student’s t test) (B and D). Bar, 10 μm (A and C).
Fig 8.
ALPCs function as intermediate docking sites for importin α/β-mediated import complexes during nuclear import.
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.
Fig 9.
CRM1-mediated export complexes accumulate at ALPCs when the disassembly of these export complexes is inhibited by transient expression of RanQ69L mutant.
(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.
Fig 10.
RanBP2 is required for the localization of RanGTP and CRM1 at NPCs and ALPCs.
(A) HeLa cells were first transfected with control or RanBP2-specific siRNAs for 48 h and then the constructs encoding FLAG-tagged RanQ69L for 24 h followed by immunofluorescence microscopy with antibodies specific to RanBP2 and FLAG. (B) HeLa cells were transfected with control or RanBP2-specific siRNAs for 72 h and then analyzed by immunofluorescence microscopy with antibodies specific to RanBP2 and CRM1. In RanBP2 RNAi cells (lower panels), “-” indicates the cell with depletion of RanBP2, and “+” indicates the cell with levels of RanBP2 that are similar to those in control cells (upper panels). The boxes at the bottom corner of each image show the enlarged version of inlets. Bar, 10 μm.
Fig 11.
A model shows how ALPCs affects nuclear import and export in the cytoplasm.
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.