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. Most functions of Ran are mediated by its 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 direction and is also a source of energy for Ran-regulated transport of cargos carried by NTRs through the channels of nuclear pore complexes. In mitosis, diffusion limited RanGTP gradient induces localized release of spindle assembly factors (SAFs) from their inhibitory complexes with nuclear import receptors, importins. As a result, SAFs are preferably activated in mitotic cytoplasm surrounding chromosomes, providing essential spatial bias to mitotic spindle assembly. However, at least some SAFs are regulated by RanGTP in mitosis with no requirement for the existence of spatially resolved RanGTP gradient. Ran-regulated SAFs are well known as cancer-related factors: TPX2, HURP, TACC3, survivin, APC and others. Focusing on the analysis of cancer cell-specific alterations of Ran-regulated mitotic mechanisms, we developed methods to of Ran function using fluorescence lifetime imaging microscopy (FLIM) of FRET biosensors expressed in live mitotic cells. Our FRET sensors called RBP-4 measures directly the RanGTP gradient and another, called Rango-4, measures the RanGTP-induced gradient of free importin beta cargos, corresponding to the gradient of activated SAFs. Using these sensors we measured the two gradients in variety of human somatic cells, including normal primary cells, immortalized cells, tumor-derived and tumor-inducing cancer cells. We found that while a steep mitotic RanGTP gradient was expressed in rapidly proliferating immortalized and/or cancer-derived human somatic cells, the gradient was strongly reduced or not detectable in slow growing human primary cells. We found that increased expression of RCC1 and large chromosomal gain are the key drivers of steep mitotic RanGTP gradients (Hasegawa et al., J. Cell Biol., 200(2)151-6, 2013). To analyze the mechanism responsible for the chromosome gain-driven rise of mitotic RanGTP gradients, we set up collaboration with the laboratory of Dr. D. Odde (University of Minnesota). In this study we use data derived from live cell measurements and computational modeling to test the hypothesis that reduced diffusion owing to chromosomal crowding is sufficient to drive steep mitotic RanGTP gradient in cells with increased chromosome number. In another follow-up to the screen for mitotic RanGTP gradients in somatic cells, we found that the in vitro-induced transformation of normal somatic cells into precursors of cancer stem cells (CSCs) is accompanied by a dramatic increase of mitotic RanGTP gradient and increased RCC1 expression. Because we observed such activation of RanGTP production during the induction of pluripotency by the Yamanaka factors, we hypothesize that increased RCC1 expression and steep RanGTP gradients are required for the maintenance of de-differentiated state of CSCs.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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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
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
Kaláb, Petr; Soderholm, Jon (2010) The design of Förster (fluorescence) resonance energy transfer (FRET)-based molecular sensors for Ran GTPase. Methods 51:220-32