QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 June 2008
doi: 10.1242/jcs.024091

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lin, J. Articles by Huang, J. J. Search for Related Content PubMed PubMed Citation Articles by Lin, J. Articles by Huang, J. J. Social Bookmarking                         What's this?

Nucleolar localization of TERT is unrelated to telomerase function in human cells

Jian Lin^*, Rui Jin^*, Bin Zhang^*, Hao Chen^*, Yun Xiu Bai, Ping Xun Yang, Su Wen Han, Yao Hua Xie, Pei Tang Huang, Cuifen Huang and Jun Jian Huang

Laboratory of Tumor and Molecular Biology, Beijing Institute of Biotechnology, 27 Taiping Road, Beijing, People's Republic of China

View larger version (12K): [in this window] [in a new window]   Fig. 1. Subcellular distributions of TERT C-terminal polypeptide sequences expressed in HeLa cells. (A) Schematic presentation of full-length TERT and its indicated C-terminal polypeptide fragments used in this study. (B) An example of the subcellular distribution patterns of different TERT C-terminal fragments in transiently transfected HeLa cells (refer to text for detailed description). 400x magnification. Green, signal for ectopically expressed GFP-tagged fragments; red, signal for the co-expressed RFP-B23; blue, DAPI nuclear staining. The merge shows the overlay images of GFP-TERT fragments and the co-expressed RFP-B23 nucleolar protein within nuclei. The percentages of cells with the represented GFP staining patterns in the corresponding transfected cell populations are shown to the right (>400 expressing cells counted for each group).

View larger version (30K): [in this window] [in a new window]   Fig. 2. Residues 965-981 of the TERT polypeptide are an active NTS sequence. (A) Alignment analysis shows that the sequence aa965-981 of TERT is highly conserved in its counterparts from mouse and Xenopus, and that it is enriched with positively charged residues. (B) Substitution of the three conserved positively charged residues indicated in A with alanines abolished nucleolar localization of the fragment GFP-TERTaa965-1132 in both human (HeLa) and mouse (NIH3T3) cells. Arrows indicate nucleoli. (C) Fusion of the aa965-981 sequence of TERT to GFP leads to nucleolar accumulation of the expressed fusion protein in both HeLa and NIH3T3 cells. The fluorescence signal shows the localization patterns of the GFP fusion proteins in their corresponding transfected cells. Arrows indicate nucleoli that were visualized by using an optic microscope. 400x magnification. The percentage of cells with the represented GFP staining patterns in the corresponding transfected cell populations are shown to the right (>400 expressing cells counted for each group).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Mutational inactivation of the C-terminal NTS of TERT completely disrupts TERT nucleolar localization in both normal and malignant human cells. Wild-type GFP-TERT and the mutant GFP–TERT-3A, which contains the substitution mutations indicated in Fig. 2A, were individually introduced, through recombinant lentiviral infection, into BJ cells (normal human fibroblasts) or into HeLa, H1299 or U2OS cancerous cells for stable expression. (A) A representative example showing that ectopically overexpressed GFP-TERT (green, upper panel) concentrates within nuclear foci identical to the nucleolar structures observed under optic microscope observation. However, overexpressed GFP–TERT-3A (green, lower panel) distributed diffusely in the nucleoplasm, outside nucleoli, in stable BJ cells. 100x magnification. (B) Images showing that overexpressed GFP-TERT exhibited a predominant nuclear diffusion pattern in all indicated transfected cancer cell lines and that GFP–TERT-3A displayed a predominant nucleolar-exclusion pattern in these cell lines. 400x magnification. Arrows indicate nucleolar regions identified under optic microscope observation. The percentages of cells with each representative image in the corresponding cell populations are presented to the right.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Mutational inactivation of the C-terminal NTS of TERT abrogates the DNA-damage-induced nucleolar localization of TERT. Wild-type GFP-TERT and the mutant GFP–TERT-3A were stably expressed in the indicated human cancer cell lines through lentiviral infection. (A) Treatment with 50 µM etoposide for 6 hours stimulated the expressed GFP-TERT to translocate from the nucleoplasm to nucleoli in all tested stable cancer cell lines. (B) The same etoposide treatment did not alter the intranuclear distribution of the expressed GFP-TERT mutant in all tested stable cells. Green represents the fluorescence signal for expressed GFP-TERT or GFP–TERT-3A in the corresponding stable cells before and after etoposide treatment. Arrows indicate the nucleolar structures observed under the optic microscope. 400x magnification. The percentage of cells with the represented GFP staining patterns in the corresponding transfected cell populations are shown on the right (>400 expressing cells counted for each group).

View larger version (61K): [in this window] [in a new window]   Fig. 5. Mutational disruption of TERT nucleolar localization does not affect its telomerase enzymatic activity in both normal and cancerous human cells. BJ cells (at around 45 PDs in culture) and U2OS cells were infected with recombinant lentivirus expressing GFP-TERT or GFP–TERT-3A. (A,C) Western-blotting assay with specific anti-GFP antibodies demonstrated that the stable expression of wild-type GFP-TERT and the mutant GFP–TERT-3A was at approximately the same level in corresponding stable BJ (A) and U2OS (C) cells. (B,D) TRAP assay demonstrated that stable expression of both GFP-TERT and of the GFP–TERT-3A mutant can activate telomerase activity with the same efficiency in both BJ fibroblasts (B) and in cancerous U2OS cells (D). Tubulin was used as the control to show the amounts for each loading sample in the western-blotting assays. Vector-infected BJ cells and U2OS cells were used as controls for these experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Mutational disruption of TERT nucleolar localization does not affect its biological effects on maintaining telomere-length and extending replicative life-span in BJ fibroblasts. (A) Telomere lengths of corresponding stable BJ cells were analyzed at the indicated time points and showed that the telomere length of vector-infected BJ cells underwent a progressive shortening with the process of cultural propagation, whereas the telomere lengths both of GFP-TERT- and GFP–TERT-3A-expressing BJ cells were stably maintained during their successive propagation in culture. (B) Growth-curve analysis showed that vector-infected BJ cells ceased proliferation at around 65 PDs, whereas both GFP-TERT- and GFP–TERT-3A-expressing BJ cells still retained a robust proliferation rate at 92 and 86 PDs, respectively. (C) SA-β-gal assay showed that vector-infected BJ cells at 65 PDs became morphologically enlarged and displayed strong positive results of SA-β-gal staining, whereas both GFP-TERT- and GFP–TERT-3A-expressing BJ cells at the indicated time points were only faintly stained by the SA-β-gal assay and were without morphological changes. 100x magnification.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter