Dissection of Mitotic Ran Pathway Function Using the Small Molecule Importazole

The faithful and proper segregation of the genome between dividing cells is of paramount importance to all organisms. In order to maintain the integrity of the genetic information during division, cells make use of an extremely complex and highly regulated set of processes known collectively as mitosis. A key aspect of mitosis is the generation and maintenance of the mitotic spindle, a large and complex microtubule based structure that is responsible for organizing and transporting the chromosomes to the daughter cells. The mitotic spindle is of vital importance to the life of the cell, and multiple partially redundant pathways have evolved to regulate its assembly and operation. The small GTPase Ran governs one such pathway functioning in the vicinity of the chromosomes to control the activation of a variety of proteins that contribute to mitotic spindle assembly. However, in addition to promoting spindle assembly, the Ran pathway also regulates a number of other essential processes during the cell cycle such as nucleocytoplasmic transport and nuclear envelope dynamics. Due to this fact, studying the mitotic roles of the Ran pathway in vivo is challenging, as mitosis comprises only a small portion of the cell cycle. In order to overcome this obstacle, we took a small molecule inhibitor based approach, and sought to identify a compound capable of disrupting Ran pathway function with great temporal precision in living cells. In the following dissertation, we first provide an introduction to the cellular processes regulated by mitotic Ran pathway function, and then follow with descriptions of our efforts to develop and improve an inhibitor of RanGTP/importinnb function. Finally, we describe how we made use of this inhibitor to gain insight into a newly discovered role for Ran in the regulation of mitotic spindle positioning.First we set out to develop a small molecule inhibitor capable of disrupting Ran pathway function. During interphase, the transport receptor importinnb carries cargoes into the nucleus, where RanGTP releases them. A similar mechanism operates in mitosis to generate a gradient of active spindle assembly factors around mitotic chromosomes. We implemented a FRETnbased, highnthroughput small molecule screen for compounds that interfere with the interaction between RanGTP and importinnb and identified importazole, a 2,4ndiaminoquinazoline. We found that importazole specifically blocked importinnbnmediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1nmediated nuclear export. When added during mitosis, importazole impaired the release of an importinnb cargo FRET probe and caused both predicted and novel defects in spindle assembly. Together, our results identified importazole as a compound suitable for study of the Ran pathway in mitosis that specifically inhibits importinnb function, and suggest a possible molecular mechanism for importazole in which it alters importinnb interaction with RanGTP.With an inhibitor of the pathway in hand, we attempted to improve importazole as a tool for study of mitotic Ran pathway function by elucidating its mechanism of action and devloping more potent analogues to maximize compound specificity. In order to gain further insight into importazole's molecular mechanism, we made use of surface plasmon resonance to directly measure the in vitro association between RanGTP and importinnb in the presence of importazole. In concordance with our previous observations, these experiments suggested that importazole does not destabilize the RanGTP/importinnb complex. However, the data was ultimately not reproducible enough to provide additional information about importazole function. In an effort to produce more potent inhibitors of RanGTP/importinnb function, we developed small molecule analogues based on the structure of importazole. One of these second generation compounds was capable of disrupting nucleocytoplasmic transport and mitotic spindle assembly, though it was not shown to be a significantly more potent inhibitor than importazole. Thus, we determined that importazole remains the best currently available tool for study of the Ran pathway in mitosis.Finally, we took advantage of importazole to explore RanGTP/importinnb involvement in regulating mitotic spindle positioning, a mitotic function of the Ran pathway that has only recently been discovered. Proper positioning of the spindle is required to ensure correct segregation of the chromosomes during mitosis, and is mediated through pulling forces exerted on the astral microtubules by dynein/dynactin complexes linked to the cell cortex with Gai, LGN, and the importinnb cargo protein NuMA. We found that importazole treatment disrupted mitotic spindle positioning in living cells without preventing formation of astral microtubules, and that it affected the cortical localization of both LGN and NuMA. These results demonstrated a role for RanGTP/importinnb function in spindle positioning, and our data suggest a model in which Ran may control this process through regulation of the stability of cortical positioning factors. A great deal remains to be learned about the role of the Ran pathway in mitotic spindle positioning, but importazole provides a promising avenue of study for this and other Ran mediated cellular processes.