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. At present, 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. During 2009/10, in both areas we succeeded in developing reagents and techniques which are needed to carry the key experiments. In the first approach, we made a significant progress in developing a much improved FRET sensor for quantitative fluorescence lifetime imaging microscopy (FLIM) measurements of RanGTP gradient in live cells. This sensor, called Rango-3, displays about 2.5-fold increased sensitivity in live cell measurements of RanGTP gradient compared with our previously published Rango sensors. For the first time, with Rango-3 we are now able to quantitatively monitor the changes of RanGTP-regulated importin beta cargo gradient during the course of live cell division. Unexpectedly, these experiments revealed significant attenuation of RanGTP-regulated release of importin beta cargos during the reformation of nuclear envelope, and RanGTP gradient-independent first rounds of nuclear import in nascent nuclei in HeLa cells. Using Rango-3 and live FLIM/FRET measurements, we are now setting up experiments to quantitatively compare RanGTP regulated importin beta cargo gradients in normal and transformed tissue culture cells. In the second approach, we significantly improved our method to isolate highly purified RanGTP-induced microtubules from X. laevis egg extracts for proteomic analysis. The major reason for using this system in our project is the conservation of Ran-regulated mitotic pathways. In addition, the completed sequencing of highly similar X. tropicalis genome enables efficient application of high throughput proteomic approaches. Until recently, the major limitation in using X. laevis egg extract system for proteomic analysis were the variability between batches of eggs and the need to initiate the purification of spindles from fresh extracts. We solved this problem by developing a protocol yielding extracts which survive storage at -80C without loss of activity, and by optimizing our method of capturing spindle structures on magnetic beads coated with microtubule-binding proteins. Finally, in collaboration with the laboratory of Dr. J. Yates at Scripps institute we are now ready to carry the proteomic analysis of Ran-induced spindle assembly. In addition to the research performed at NCI, we continued in collaborative projects with several extramural laboratories. In collaboration with Professor Alexey Khodjakov (Wadsworth Center, Albany, NY), we showed that RanGTP gradient functions in parallel with kinetochore-dependent mechanisms to support bipolar spindle assembly in HeLa (J. Cell Biol., 2009, 187:43). In collaboration with Professor Iris Meier (The Ohio State University, Columbus, OH), we introduced Rango FRET sensor into transgenic A. thaliana plants, which allowed us to obtain first insights into the regulation of Ran in plant cells in vivo. Finally, in collaboration with Dr. Inke Nathke (University of Dundee, UK), we described and analyzed the RanGTP- and importin beta-regulated function of APC (Adenomatous polyposis coli) in mitotic microtubule assembly (J. Cell Sci, 2010, 123:736).

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
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