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First published online 19 December 2006
doi: 10.1242/jcs.03345

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A B23-interacting sequence as a tool to visualize protein interactions in a cellular context

Institut Jacques Monod, UMR 7592 CNRS/Universités Paris 6 et 7, 2 Place Jussieu, 75251 Paris Cedex 05, France

View larger version (44K): [in this window] [in a new window]   Fig. 1. A synthetic NoLS targets GFP to nucleoli. (A) Schematic representation of the generation of NoLS. The upper diagram corresponds to the DNA sequence encoding human sirtuin-1, the middle one to the PCR product after misreading of the DNA polymerase and the bottom one to the resulting mutant protein. A partial nucleotide sequence of sirtuin-1 is indicated in the upper diagram, and the corresponding misread sequence is indicated in the middle diagram. The resulting mutant protein corresponds to the first 87 amino acids of sirtuin-1 (sirtuin-1) followed by a synthetic sequence of 93 amino acids (NoLS). The numbers are nucleotide or amino acid positions. (B) NoLS with the stretch of basic residues in red. (C) Constructs of NoLS containing the entire or partial basic stretch in red. (D) Fluorescence microscopy observations of the NoLS constructs fused to GFP and expressed in HeLa cells. Only the constructs possessing the stretch of basic residues were predominantly (NoLS/31-63, i,j and NoLS/1-50, e,f), if not exclusively (NoLS, a,b and NoLS/33-93, c,d) localized in nucleoli, whereas the other constructs were not (NoLS/51-93, k,l) or only slightly (NoLS/1-32, g,h) enriched in nucleoli. Arrowheads in panel b represent nucleoli visible by phase-contrast image. Bar, 10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 2. NoLS interacts directly with B23; this interaction involves the acidic stretches of B23. (A) GST and GST-NoLS pull-down assays performed using whole HeLa cell extracts. A sample (1%) of the extract used for each pull-down assay (Input), 1% of the supernatant (S) and 1.5% of the proteins eluted from the bead pellet (P) after the indicated pull-down assay were electrophoresed and immunoblotted to detect B23, Nopp140, Nucleolin and Nop52. B23 interacts specifically with GST-NoLS but not with GST. By contrast, Nopp140, Nucleolin and Nop52 do not interact with GST-NoLS. (B) GST and GST-NoLS pull-down assays performed using a purified recombinant B23. A sample (2%) of the supernatant (S) and 3% of the proteins eluted from the bead pellet (P) after the indicated pull-down assay were electrophoresed and immunoblotted to detect B23. B23-NoLS interaction is direct. (C) Pull-down assays performed on whole extracts prepared from HeLa cells transiently transfected with B23 wild-type (B23 wt) and B23 mutants fused to the C-terminus of GFP or DsRed as indicated. The mutants B23/121-186, B23/1-119, B23/1-132, B23/1-160, B23/1-186 and B23/187-294 correspond to the B23 mutants possessing the B23 residues 121-186, 1-119, 1-132, 1-160, 1-186 and 187-294, respectively. Black boxes on the schematic representations correspond to the stretches of acidic amino acids present in the B23 sequence. A sample (1.5%) of the extracts used for each pull-down assay (Input) and 3% of the proteins eluted after the GST-NoLS pull-down assay (GST-NoLS) were electrophoresed and immunoblotted using anti-B23 or anti-GFP antibodies. Only B23 mutants possessing at least one stretch of acidic amino acids interact with GST-NoLS. Asterisks represent the endogenous B23 revealed only with the anti-B23 antibodies but not with the anti-GFP antibodies. Arrowheads point to the overexpressed tagged-B23 or mutant B23.

View larger version (22K): [in this window] [in a new window]   Fig. 3. NoLS possesses a stronger affinity for B23 than does HIV-1 Rev. Pull-down experiments were performed using GST, GST-NoLS and GST-Rev on the same whole HeLa cell extract for increasing incubation times (1.5, 3 and 20 hours) and the binding of B23 was analyzed. (A) A sample (1.5%) of the proteins eluted after each pull-down assay was electrophoresed and analyzed by Coomassie Blue staining. No significant loss of GST, GST-NoLS or GST-Rev occurred during the pull-down assays. (B) A sample (1.5%) of the extract used for each pull-down assay (Input) and 1.5% of the proteins eluted after the indicated pull-down assay were electrophoresed and immunoblotted using anti-B23 antibodies. The signals corresponding to B23 were quantified and are expressed as a percentage of B23 present in the Input. By contrast to GST, both GST-NoLS and GST-Rev bound B23, but B23-NoLS interaction occurred more rapidly and more efficiently than B23-Rev interaction.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Localization of GFP-NoLS in HeLa cells during the cell cycle. (A) HeLa cells were transfected with GFP-NoLS fusion and analyzed by fibrillarin or B23 immunolabeling (a,e), GFP-NoLS observation (b,f) and phase-contrast (d,h). The nucleolar localization of NoLS in interphasic cells was demonstrated using fibrillarin, a marker of the DFC, and B23 preferentially localized to the GC of nucleoli. GFP-NoLS did not colocalize with fibrillarin as observed by comparing the enlargements (a-c, insets), whereas it colocalized with B23 as observed by comparing the enlargements (e-g, insets). (B) HeLa cells cotransfected with GFP-NoLS and DsRed-B23 were synchronized in mitosis. GFP-NoLS observations (a,e,i), DsRed-B23 observations (b,f,j) and superimpositions of both labelings in the same mitotic cells (c,g,k). The mitotic stage was determined by DAPI staining (d,h,l). GFP-NoLS colocalized with DsRed-B23 at the chromosome periphery in metaphase (a-d) and in numerous foci, the PNBs, in telophase (e-h) and early G1 (i-l). In early G1, GFP-NoLS and DsRed-B23 colocalized in the reformed nucleoli. These nucleoli are indicated by arrowheads (i-k). Bars, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 5. NoLS still colocalizes with B23 after actinomycin D or roscovitine treatment and is delocalized from nucleoli to nucleoplasm when the endogenous B23 is selectively delocalized from nucleoli to nucleoplasm. (A) HeLa cells cotransfected with GFP-NoLS and mDsRed-B23 or with GFP-NoLS and fibrillarin-RFP were treated with actinomycin D (a-h), or roscovitine (i-p). After actinomycin D treatment, NoLS colocalized with B23 in the segregated nucleolus (a-d) but was excluded from the fibrillarin-containing caps (e-h). After roscovitine treatment, NoLS colocalized with B23 in large nuclear bodies (i-l) but not with fibrillarin (m-p). (B) NIH-3T3 cells were transfected with GFP-B23-NES and analyzed by B23 immunolabeling (a-c) or cotransfected with GFP-B23-NES and DsRed-NoLS (d-f). After leptomycin B treatment, GFP-B23-NES was located in the nucleus and mostly excluded from nucleoli (a, arrowheads). Anti-B23 antibodies, revealing both endogenous and mutated B23, labeled the nucleoplasm and only slightly the nucleoli of GFP-B23-NES-expressing cells, in contrast to what was observed in cells not expressing GFP-B23-NES in which B23 labeling was mostly restricted to nucleoli (b). Thus, the endogenous B23 was mostly excluded from nucleoli in NIH-3T3 cells expressing GFP-B23-NES. In cells expressing GFP-B23-NES (d), DsRed-NoLS (e) was also mostly delocalized from nucleoli to nucleoplasm. Arrowheads point to nucleoli. Bars, 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 6. Fusion of NoLS to fibrillarin makes it possible to delocalize fibrillarin from the DFC to the GC of nucleoli; this mislocalization of fibrillarin in the GC of nucleoli induces the mislocalization of the fibrillarin partner Nop56 in the GC but does not affect the localization of Nopp140. HeLa cells were cotransfected with fibrillarin-GFP and DsRed-B23 (a-d), with fibrillarin-NoLS-GFP and DsRed-B23 (e-h), with fibrillarin-GFP and DsRed-Nop56 (i-l), with fibrillarin-NoLS-GFP and DsRed-Nop56 (m-p), with fibrillarin-RFP and GFP-Nopp140 (q-t) or with fibrillarin-NoLS-RFP and GFP-Nopp140 (u-x). Fibrillarin (a) localized in the DFC of nucleoli, whereas B23 (b) localized preferentially in the GC as evidenced by the superimposition of both labelings (c) and by the comparison with the phase contrast image (d). When fused to NoLS, fibrillarin (e) was delocalized from the DFC to the GC of nucleoli and therefore colocalized with B23 (f) as evidenced by the superimposition of both labelings (g). Fibrillarin (i) colocalized with Nop56 (j) in the DFC of nucleoli as evidenced by the superimposition of both labelings (k). The delocalization of fibrillarin in the GC of nucleoli by the fusion of the NoLS (m) similarly modified the nucleolar localization of Nop56 (n), and therefore both proteins still colocalized as demonstrated by the superimposition of both labelings (o). Fibrillarin (q) and Nopp140 (r) colocalized in the DFC of nucleoli as evidenced by the superimposition of both labelings (s). By contrast to Nop56, the delocalization of fibrillarin fused to NoLS (u) did not modify the nucleolar localization of Nopp140 (v) as shown by the superimposition of both labelings (w). Bar, 10 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 7. The fusion of NoLS to MafG makes it possible to delocalize the nuclear complex MafG-P45 to nucleoli. HeLa cells were cotransfected with GFP-P45 and DsRed-B23 (a-d), with DsRed-MafG and GFP-B23 (e-h), with DsRed-NoLS-MafG and GFP-B23 (i-l), with DsRed-NoLS-MafG and GFP-P45 (m-p), with DsRed-NoLS and GFP-P45 (q-t) or with DsRed-NoLS-MafG and GFP-sirtuin-1 (u-x). P45 (a) and MafG (e) were localized in the nucleus and excluded from the nucleoli as observed by comparing with B23 localization (compare a,b and e,f) and by the superimpositions of labelings (c,g). When fused to NoLS, MafG (i) colocalized with B23 (j) in nucleoli as evidenced by the superimposition of both labelings (k). Targeting of MafG to nucleoli by addition of the NoLS induced a clear relocalization of P45 to the nucleoli (compare m,n,o and the phase contrast image, p). Coexpression of DsRed-NoLS and GFP-P45 (q-t) showed that the expression of NoLS did not modify the nuclear localization of P45. By contrast to P45, targeting of MafG to nucleoli by the addition of NoLS (u) did not modify the nuclear localization of sirtuin-1 (v). Bar, 10 µm.

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