中間絲波紋蛋白在細胞,端粒維持和細胞不死化中的作用

1、Potential role of the intermediate filament protein vimentin in telomere maintenance and spontaneous cell immortalization. Jian-Ping Lu, Genrich V. Tolstonog and Peter TraubAbstractSimilar to knockout of telomerase RNA, knockout of the vimentin gene severely impairs spontaneous immortalization of pr

2、imary mouse embryo fibroblasts. Therefore, the expression and activity of telomerase in primary and immortalized embryo fibroblasts from wild-type (Vim+/+) and vimentin knockout (Vim-/-) mice were studied. While the primary Vim+/+ fibroblasts exhibited telomerase activity and progressively reduced t

3、heir growth potential during serial transfer, passed through a growth minimum around passage 12 and, as immortalized cells, resumed faster growth, the primary Vim-/- fibroblasts did not show detectable telomerase activity, cut down their growth rate much earlier and immortalized at an extremely low

4、rate. In primary Vim-/- cells at the growth minimum, telomere chromatin was strongly decondensed and mainly associated with the nucleoli, in contrast to primary Vim+/+ cells in which the telomeres were less decondensed and localized predominantly at the nuclear periphery. Although the telomerase act

5、ivity was significantly lower or even undetectable in several immortalized Vim-/- cell clones in comparison to that of immortalized Vim+/+ clones, there were no differences between telomere length in primary cells and telomere shortening associated with serial passage of immortalized cells. Despite

6、the absence of telomerase activity from Vim-/- cell clones, these did not show any signs of impaired growth after a large number of cell passages (550-560 at the endpoint of the experiment). Due to the up-regulated expression of proteins involved in homologous (Rad51, Rad52) and non-homologous end-j

7、oining (Ku70, Ku80) recombination in Vim-/- clones, in these cells probably a telomerase-independent, alternative lengthening of telomeres (ALT) mechanism on the basis of DNA recombination is operative. Analysis of telomerase reverse transcriptase (TERT) gene transcription revealed a complex pattern

8、 of alternative splice and transcription start products varying between Vim+/+ and Vim-/- cells. The alternative forms of TERT are probably enzymatically inactive and might fulfill a key function in turning on the recombination-dependent telomere lengthening pathway. Employing immunostaining of TERT

9、 in telomerase-positive cells extracted with Triton X-100 and treated with Benzonase before fixation, the TERT as well as TRF2 and TP1 telomere-binding proteins were found associated with the nuclear lamina. Applying a co-immunoprecipitation assay to cell lysates, direct interaction of vimentin with

10、 catalytically active telomerase RNP was demonstrated. The telomerase activity in Vim-/- cell lysates was slightly stimulated, but significantly inhibited in Vim+/+ cell lysates after the addition of exogenous vimentin. The -helical rod domain of vimentin was shown to exert the inhibitory effect on

11、telomerase activity in Vim+/+ cell lysates and is probably responsible for the binding of vimentin to telomerase RNP. In addition, employing the chromatin immunoprecipitation (CHIP) protocol, vimentin was found associated with telomere DNA. Our results suggest a direct involvement of vimentin in the

12、 maintenance of telomeres and in spontaneous cell immortalization. Monolayers of Vim+/+ and Vim/ fibroblasts were subjected to serial passaging by splitting them at a ratio of 1:2 on a 3.5 day transfer regime. Among the residual Vim/pr cells of transfer 10, at most one or two cells immortalized and

13、grew out into colonies at an extremely low rate, whereas about 15% of the Vim+/+pr fibroblasts passed through growth crisis at passage 11/12 and thereafter proliferated at increasing rates.Vim+/+ ( ) fibroblasts Vim / (o) fibroblastsgrowth minimumspontaneousimmortalizationprimarycell cultureFigure 1

14、. Rate of spontaneous immortalization of the vimentin-positive and vimentin-negative mouse embryo fibroblastsTolstonog GV, Shoeman RL, Traub U and Traub P. Role of the intermediate filament protein vimentin in delaying senescence and in the spontaneous immortalization of mouse embryo fibroblasts. DN

15、A and Cell Biology, 2001, V. 20, pp. 509-529 Vimentin Positive (Vim+) Vimentin Negative (Vim- ) Cells Passage* Cells Passage* Vim+/+ 10-12 Vim-/- 10-12 clones: clones: A4 low III7E low A4 high III7E high B7 low III8F low B7 high III8F high IV2E low IV2E high IV8B low IV8B high *low = passage 20-30,

16、high = passage 200-500; cells below passage 10-12 (“crisis”) are primary cells (pr), those above passage 12 are spontaneously immortalized (im) cells. Immortalized vimentin-positive and vimentin-negative cells were expanded into the clones by limiting dilution method.prprTable 1. Vimentin-positive a

17、nd vimentin-negative mouse embryo fibroblastsGeneration time (hours)020406080100Col 1 Plot 1 ZeroFigure 2. Cell doubling rate of spontaneously immortalized vimentin-positive and vimentin-negative mouse embryo fibroblastsvimentin-negative cellsvimentin-positive cellsA4B7III7EIII8FIV2EIV8B L H L H L H

18、 L H L H L HL low passageH high passageTolstonog GV, Belichenko IV, Lu J.-P, Hartig R, Shoeman RL, Traub U and Traub P. Spontaneously immortalized mouse embryo fibroblasts: growth behavior of wild-type and vimentin-deficient cells in relation to mitochondrial structure and activity. (in preparation)

19、Lu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filament protein vimentin in telomere maintenance. (in preparation) ADBECFFigure 3. Telomere chromatin organization in the vimentin-positive and vimentin-negative mouse embryo fibroblastsVimpr-/-, tr. 8Vimpr+/+, tr. 10Vimpr-/-, tr

20、. 9 (crisis)Vimim-/-, tr. 8Vimpr+/+, tr. 11Vimpr+/+, tr. 12relative telomerase activity +STD0,00,20,40,60,81,01,21,41,61,8vimentin-negative cellsvimentin-positive cellsA4B7III7EIII8FIV2EIV8BVimpr+/+Vimpr-/-A4B7 III7EIII8FIV2EIV8B97 kb45 kb23 kb9 kb6 kb4 kbFigure 4. Telomere length and telomerase act

21、ivity in the immortalized vimentin-positive and vimentin-negative mouse embryo fibroblastsTelomere restriction fragment analysisTelomerase activity assayLu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filament protein vimentin in telomere maintenance. (in preparation) Vim+/+prV

22、im / prA4III7ELHLHtelomerase activity+-+-expression:mTERTExon2-spATG201other products-+?-+?+?+?+-?-?Lu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filament protein vimentin in telomere maintenance. (in preparation) Figure 5. TERT gene expression products in the immortalized vi

23、mentin-positive and vimentin-negative mouse embryo fibroblastsNested PCR analysis of TERT gene expressionLu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filament protein vimentin in telomere maintenance. (in preparation) Figure 6. Proteins involved in the ALT pathway in the imm

24、ortalized vimentin-positive and vimentin-negative mouse embryo fibroblasts020406080100B7A4III7EIII8FIV2EIV8Bvimentin-negative cellsvimentin-positive cells010203040506070B7A4III7EIV2EIV8Bvimentin-negative cellsvimentin-positive cellsIII8FKu70Ku8001020304050B7A4III7EIII8FIV2EIV8Bvimentin-negative cell

25、svimentin-positive cellsRad52020406080MLH1B7A4III7EIII8FIV2EIV8Bvimentin-negative cellsvimentin-positive cellsLu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filament protein vimentin in telomere maintenance. (in preparation) Figure 7. Non-homologous end joining activity in the

26、 immortalized vimentin-positive and vimentin-negative mouse embryo fibroblasts TERT lamin B DAPIvim+ A4vim- III7EFigure 8. Association of telomerase reverse transcriptase (TERT) with nuclear lamina in the CSK-extracted cells.Lu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filam

27、ent protein vimentin in telomere maintenance. (in preparation) Relative telomerase activity0,00,20,40,60,81,01,21,41,6Col 3 Plot 1 Zero0,00,20,40,60,81,01,21,41,6cell lysate - + + + + - + + + +vimentin - + - - - - + - - -antibody - + + - - - + + - -beads + + + + - + + + + -Vim+/+ im (passage 520)vim

28、- IV8B (passage 520)Lu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filament protein vimentin in telomere maintenance. (in preparation) Figure 9. Co-immunoprecipitation of the active telomerase with vimentin.0100250500100020000,00,20,40,60,81,01,21,41,60100250500100020000,00,20

29、,40,60,81,01,21,41,60100250500100020000,00,20,40,60,81,01,21,41,61,80100250500100020000,00,20,40,60,81,01,21,41,60100250500100020000,00,20,40,60,81,01,21,41,6 0100250500100020000,00,10,20,30,40,50,6vimentin (ng)CP-vimentin (ng)actin (ng)vimentin (ng)vimentin (ng)actin (ng)Vim+/+ im (passage 520)vim- IV8B (passage 523)Vim+/+ im (passage 520)Vim+/+ im (passage 520)vim- IV8B (passage 523)vim- IV2E (passage 30)Lu J.-P, Tolstonog GV and Traub P. Potential role of the intermediate filament protein vimentin in telomere maintenance. (in prepara