Potential role of the intermediate filament protein vimentin in telomere maintenance and spontaneous cell immortalization. Jian-Ping Lu, Genrich V. Tolstonog and Peter Traub Abstract Similar to knockout of telomerase RNA, knockout of the vimentin gene severely impairs spontaneous immortalization of primary 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 their 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 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 activity 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 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-joining (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 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 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 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 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 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 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) fibroblasts growth minimum spontaneous immortalization primary cell culture Figure 1. Rate of spontaneous immortalization of the vimentin-positive and vimentin-negative mouse embryo fibroblasts Tolstonog 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. DNA 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, 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. prpr Table 1. Vimentin-positive and vimentin-negative mouse embryo fibroblasts Figure 2. Cell doubling rate of spontaneously immortalized vimentin-posit