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, 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. We are focusing on the role of Ran in mitotic spindle assembly and our goal is to elucidate differences 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: 1) The analysis of cancer cell-specific alterations of Ran-regulated mitotic mechanisms2) Biochemical and functional analysis of Ran-regulated mitotic pathways In the first approach, we developed methods for highly sensitive quantitative measurements of Ran function in mitotic cells using fluorescence lifetime imaging microscopy (FLIM) of FRET biosensors expressed in live cells. One of 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, in 2010-12 we measured the two gradients in a panel of 14 different 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 human somatic cells, including normal breast epithelial MCF10a cells and cancer-derived HeLa cells, the gradient was strongly reduced or not detectable in slow growing human primary cells, such as HFF-1 fibroblasts. Consistent with the role of RanGTP in accelerating the spindle assembly, disruptions of RanGTP gradient caused delays in prometaphase progression. In vitro cell fusion induced steep mitotic RanGTP gradient in HFF-1 cells and increased the steepness of the gradient in MCF10a and HeLa cells, indicating that chromosomal gain in aneuploid cancer cells can promote their spindle assembly via Ran. A manuscript describing our findings was reviewed by J. Cell Biol., and we are currently working on the response on mostly positive reviews. In the second approach, we focused on the mitotic spindle assembly mechanisms regulated by the complex or importin beta and its adaptor proteins of the importin alpha family. Although there are 7 importins alpha expressed in human genome, only one of them, importin alpha 1 (KPNA2) was identified as SAF regulator. Interestingly, high levels of KPNA2 expression strongly correlate with poor prognosis in several forms of cancer. We are interested to determine whether importin alpha 1 is indeed the importin alpha isoform specialized for mitotic functions and to comprehensively determine the complete list of its mitotic targets. To that end, we developed assays in Xenopus laevis egg extracts to compare the roles of different human importins alpha in mitotic spindle assembly. These assays are relevant because previous studies determined high level of functional homology between the role of Xenopus and human importins and different SAFs in mitotic spindle assembly. Surprisingly, our results so far indicate a dominant negative mutant of importin alpha 3 (KPNA4) functions as even more potent inhibitor of mitotic spindle assembly than a similar mutated form of importin alpha 1, indicating the existence of importin alpha 3 targeted SAF(s). Work is in progress to identify the importin alpha 3-binding SAFs by mass spectrometry and then to analyze their mitotic function in mitotic spindle assembly by cell biological approaches.
