Ran GTPase is a key regulator of macromolecular transport between nucleus and cytoplasm and has important role in several steps of cell division, including mitotic spindle assembly and nuclear envelope reformation at the exit from mitosis. Because RCC1, the guanine nucleotide exchange factor for Ran, binds to chromatin while RanGAP is cytoplasmic, the position of chromosomes is marked by the highest cellular concentration of RanGTP, the RanGTP gradient. The cellular functions of Ran are mediated by RanGTP interactions with importin beta-related nuclear transport receptors (NTRs). Ran and NTRs functionally interact with nucleoporins (Nups) the components of NPCs. In interphase, step-wise RanGTP gradient across nuclear envelope provides directionality to the Ran-regulated transport of cargos carried by NTRs through nuclear pore complexes. During mitosis, chromosomes are surrounded by a steep diffusional gradient of RanGTP which continues to regulate the loading and unloading of cargos on NTRs in the mitotic cytoplasm. Because some mitotic regulators are at the same time NTR cargos, their activities or binding to mitotic structures are controlled by RanGTP, contributing to mitotic spindle assembly and chromosome segregation to daughter cells. Previously, we focused on the role of Ran in mitotic spindle assembly and our goal was to elucidate differences in the contribution of Ran to mitosis in cancer cells vs. normal cells. To that end, we developed methods for quantitative measurements of Ran function in mitotic cells using fluorescence lifetime imaging microscopy (FLIM) of FRET biosensors expressed in live cells. Using this approach, we found that the mitotic RanGTP gradients were more robust in rapidly proliferating normal and cancer cells. Furthermore, we showed that increased RCC1 expression and increased number of mitotic chromosomes in aneuploid cells were the two most important factors determining the steepness of mitotic RanGTP gradients. As a follow up to this study, which was published in 2013, we pursued two approaches. First, we set out to determine the mechanisms of chromosome gain-driven activation of mitotic RanGTP gradients. We hypothesized that the underlining mechanism depends on the biophysical properties of the chromosome-cytoplasm interface in mitotic cells and involves the increased number of chromosome binding sites for RCC1 and reduced diffusion in the center of mitotic cells with increased chromosome number. To test this hypothesis, we planned combining live cell measurements with computational modeling of the mitotic RanGTP gradient, in collaboration with the laboratory of Dr. David Odde at the University of Minnesota. We performed initial sets of measurements and modified the existing computational models for our purposes. However, during 2013 Dr. Hasegawa decided to leave NIH and focus his future career on teaching physics and biophysics at college level. After a successful job search, Dr. Hasegawa accepted a teaching position at Amherst University and left the lab, as well as his unfinished project at the end of 2013. In the second approach, we studied the role of Ran in the regulation of cell cycle. In the first phase of this project we analyzed role of Ran in the exit from cell cycle in senescent cells. We found that, as a result of irreversible cytoplasmic processing of chromatin, the permanent G1/S arrest in senescent cells was associated with a strong decline of RCC1 protein levels. That result was consistent with our initial hypothesis that the depletion of RanGTP levels enforces the stable exit from cell cycle via reducing the Ran-regulated nuclear-cytoplasmic transport of cell cycle factors. This research was performed primarily by Dr. Ryu, with the help of other members of the lab. Unfortunately, Dr. Ryu's progress was compromised by a serious technical problem that prevented him to complete this work before leaving for a new job at the end of September 2013. As a result, both mainprojects in the lab were left unpublished and unstaffed about half a year before the Site Visit of the lab in June 2014. Not surprisingly, the unfavorable Site Visit review led to the recommendation of the Board of Scientific Advisors to permanently close the lab in February 2016. In the remaining time, Dr. Cekan, the remaining recently hired postdoctoral researcher, and I have focused on the cell cycle project. We obtained evidence that RCC1-driven activation of Ran accelerates the reentry to cell cycle following DNA damage, leading to the evasion of cell senescence in normal and cancer cells. In 2014, we submitted a manuscript describing those result for publication (Nature Communications) but received a negative editorial feedback that indicated a need for mechanistic insight into the Ran's function in senescence. In 2015, our progress to that end was slowed by three months absence of Dr. Cekan from the lab, for medical reasons. Nevertheless, we are currently finalizing an updated manuscript for a new submission for publication.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Hasegawa, Keisuke; Ryu, Sung Jin; Kalab, Petr (2013) Chromosomal gain promotes formation of a steep RanGTP gradient that drives mitosis in aneuploid cells. J Cell Biol 200:151-61
Halpin, David; Kalab, Petr; Wang, Jay et al. (2011) Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts. PLoS Biol 9:e1001225
Kalab, Petr; Solc, Petr; Motlik, Jan (2011) The role of RanGTP gradient in vertebrate oocyte maturation. Results Probl Cell Differ 53:235-67
Soderholm, Jonathan F; Bird, Stephen L; Kalab, Petr et al. (2011) Importazole, a small molecule inhibitor of the transport receptor importin-ýý. ACS Chem Biol 6:700-8
Kaláb, Petr; Soderholm, Jon (2010) The design of Förster (fluorescence) resonance energy transfer (FRET)-based molecular sensors for Ran GTPase. Methods 51:220-32
Dikovskaya, Dina; Li, Zhuoyu; Newton, Ian P et al. (2010) Microtubule assembly by the Apc protein is regulated by importin-beta--RanGTP. J Cell Sci 123:736-46
Lowe, Alan R; Siegel, Jake J; Kalab, Petr et al. (2010) Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking. Nature 467:600-3
O'Connell, Christopher B; Loncarek, Jadranka; Kaláb, Petr et al. (2009) Relative contributions of chromatin and kinetochores to mitotic spindle assembly. J Cell Biol 187:43-51
Abu-Arish, Asmahan; Kalab, Petr; Ng-Kamstra, Josh et al. (2009) Spatial distribution and mobility of the Ran GTPase in live interphase cells. Biophys J 97:2164-78