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, but not all, 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, some SAFs are regulated by RanGTP in mitosis with no requirement for the existence of spatially resolved RanGTP gradient. Remarkably, most of the SAFs involved in Ran-regulated mitotic network are well known as cancer-related factors: TPX2, Aurora A, hTOG, HURP, BRCA1, RHAMM, NPM1, RASSF1a, TACC3/maskin, survivin, APC (adenoma polyposis coli) and others. In addition, more recently it was shown that upregulation of RanGTP gradient leads to transformation of NIH3T3 cells apparently through causing amplification of RanGTP-gradient dependent cytoplasmic decapping of mRNAs, which and thus inducing deregulated synthesis of growth promoting functions. In summary, multiple pieces of evidence suggest that potentially several different RanGTP gradient-regulated processes have an important role in cancer etiology. We are focusing on the role of Ran in mitotic spindle assembly and our goal is to elucidate differences, if any, in the contribution of Ran to mitosis in cancer cells vs. normal cells. Many of the Ran-regulated mitotic mechanisms of spindle assembly are highly conserved between different organisms. Thus, Ran-regulated SAFs) carry similar functions in Xenopus laevis meiotic/embryonic egg extracts, in meiotic mouse oocytes and in human tissue culture cells, suggesting their evolutionary conservation. For example, TPX2 activates Aurora A in HeLa cells and in X. laevis egg extracts. However, the relative contribution to spindle assembly and cell division is dramatically between different types of cells, such as in comparison of meiotic vs. somatic cells. We use two approaches in addressing these important questions: 1) Quantitative analysis of RanGTP gradient in mitotic normal and cancer cells 2) Proteomic and functional reconstitution analysis of Ran-regulated mitotic spindle assembly. In the first approach, in 2009/10 we developed improved FRET sensors for quantitative fluorescence lifetime imaging microscopy (FLIM) measurements of RanGTP gradient in live cells. Using these sensors, in the fall of 2010 we discovered that in striking contrast to HeLa, mitotic RanGTP gradient is virtually absent in normal primary human fibroblasts. This surprising finding was confirmed by two different FRET imaging methods, each with two different FRET sensors: RanGTP- binding sensor and sensor for RanGTP-regulated importin beta cargos. Moreover, we soon also determined that metastatic breast cancer MCF10CA1a cells displayed stronger RCC1 binding to chromatin and steeper mitotic RanGTP gradient than isogenic normal or non-malignant immortalized breast cancer cell lines. Because of the potentially high significance of this finding, in 2011 we focused most of the research in the lab on investigating the molecular mechanism underlying the differences in RanGTP gradient in normal vs. cancer cells. We found addition to increased concentration of Ran, the key factor responsible for steeper RanGTP gradients was increased RCC1 binding to chromatin. Live-cells fluorescence recovery after photobleaching (FRAP) measurements with RCC1-mCherry showed that throughout the cell cycle, the binding of RCC1 to chromatin was stronger in HeLa than in fibroblasts, required N-terminal methylation by NRMT and was supported by RCC1 phosphorylation on Serine 10. While NRMT depletion caused decreased RCC1 binding to chromatin and spindle defects in HeLa, the same treatment had no significant effect on spindle assembly in normal fibroblasts. Consistent with proposed Ran functions in mitotic spindle assembly, the absence of a steep mitotic RanGTP gradient in primary cells correlated with extended prometaphase. These findings suggest that RanGTP gradient and its role in mitotic spindle assembly are attenuated in normal somatic tissues by mechanisms including decreased RCC1 methylation. On the other hand, steep mitotic RanGTP gradient is a commonly expressed hallmark of rapidly dividing normal and cancer cells. We wrote a manuscript describing these findings and plan submitting it for publication within the next few weeks (aiming for October 1, 2011).

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
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Kaláb, Petr; Soderholm, Jon (2010) The design of Förster (fluorescence) resonance energy transfer (FRET)-based molecular sensors for Ran GTPase. Methods 51:220-32
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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