A. Mitotic functions of Ran Using quantitative fluorescence lifetime imaging microscopy (FLIM) of FRET sensors we investigate the functions of the mitotic RanGTP gradient in normal and cancer-derived cells. We succeeded in building a functional time correlated single photon counting (TCSPC) FLIM system (Becker-Hickl) which is attached to Zeiss 710 confocal microscope with 2 photon excitation. To facilitate the Ran gradient imaging in relatively very small normal human fibroblast mitotic cells, we developed FLIM/FRET sensor utilizing non-fluorescent quenching acceptor sREACH. In our first tests, these sensors significantly outperformed our previous best generation of Ran sensors. Using the new Zeiss 710 system we succeeded in imaging the reorganization of Ran-regulated importin beta cargo gradient during the formation of the nuclear envelope in telophase in live HeLa cells. These results confirmed our previous set of data obtained in X. laevis egg extracts and suggested an unexpected new model of RanGTP gradient role in nuclear envelope formation. We expect that this study will be completed and submitted for publication within 3-4 months. B. Ran in senescence and cancer We examine the the role of the regulation of nuclear transport system has important function in the replication-induced cell senescence vs. the onset of cancer, both being intrinsically age-related phenomena. We hypothesized that while senescent cell program triggers overall decrease of nuclear transport system, possibly an opposite trend takes place in immortalized cancer cells. Our data on protein levels and localization obtained with young and aged normal human fibroblasts and Hela cell lines confirm this part of our prediction. We are now setting up experiments to examine the RanGTP gradient in our test cell lines and to see whether specific manipulation of the nuclear transport system could be used to either accelerate or prevent the cell senescence onset. This is an early stage of the project that started only 4-5 months ago.
