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SummaryThe cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.

The positive influence of undergraduate research and mentoring on student success in STEM fields has been well-established. However, the role that the gender of a research mentor may play in the undergraduate research experience warrants further investigation. This is an especially critical issue to address, since the lack of female role models in STEM fields is acknowledged as an impediment to the success and progress of women pursuing STEM-careers. To evaluate how the gender of undergraduate research mentors influences the research experience of students, we collected and analyzed surveys from undergraduates and alumni who had completed undergraduate research at the University of Nebraska at Kearney. We found that even though students did not select mentors based on gender, there were differences in how students perceived their mentors, depending on the gender of their mentors. Interestingly, students with female mentors were more likely than students with male mentors to report that their research experience had prepared them for a career in science. Further, our gender-pairing analyses revealed that students who expressed that the gender of their mentor had contributed to their relationship with their mentor were more likely to have a female mentor. Our data indicate that female mentors favorably influence the undergraduate research experience of both male and female students. Finally, our study reinforces the conclusions of previous studies demonstrating that undergraduate research and mentoring are beneficial for students. Overall, our findings support that, for students to fully benefit from their undergraduate research experience, undergraduate research opportunities for students should include an equitable representation of female mentors.

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A plethora of proteins and processes regulate proper mitosis and are yet to be fully understood. Since mitotic cells are an emerging target for cancer therapy, it is of utmost importance to decipher the mechanisms that regulate mitosis. The purpose of our study is to identify the role CUL3 plays in regulating mitosis. Specifically, it is our aim to identify CUL3 interacting partners that play roles in spindle assembly, in order to understand the function of CUL3 in constructing the mitotic spindle. The CUL3 protein is an ubiquitin E3 ligase scaffold. By means of ubiquitination, CUL3 can regulate an array of proteins. We have performed CUL3 RNAi experiments, followed by cell cycle analysis using flow cytometry and immunostaining of DNA, α-tubulin and γ-tubulin, to identify the importance of CUL3 in regulating mitosis. Furthermore, we have performed CUL3 immunoprecipitation (IP) experiments in X.laevis mitotic egg extracts and in HeLa cells synchronized at mitosis, to identify the binding partners of CUL3 which orchestrate mitosis. Using treatment with either nocodazole (promotes microtubule depolymerization) or monastrol (causes microtubule stabilization), we performed interaction studies in HeLa cells to determine the effect of microtubule polymerization on the interaction of CUL3 with its mitotic binding partners. Similarly, in X.laevis egg extracts, we have used warm (for microtubule depolymerization) and cold (for microtubule stabilization) IP conditions to determine the effects of microtubule status on CUL3 interaction with its binding partners. Our results have shown that CUL3-depleted cells display a dramatic mitotic phenotype which includes: defective spindles, multiple centrosomes, enlarged cells and nuclei, G2/M accumulation and delayed cytokinesis. To understand how CUL3 may participate in the formation of the mitotic spindle, we determined the binding partners of CUL3 that are involved in spindle assembly. CUL3 IP results from both X.laevis egg extracts and HeLa cells indicate that CUL3 interacts with the kinesin motor protein, Eg5. Eg5 is an important spindle assembly factor, which is essential in mitosis for proper formation of a bipolar spindle. Interestingly, targeting Eg5 is emerging as an attractive means to inhibit the proliferation of cancer cells. Thus, better characterizing the interaction of CUL3 and Eg5 is critical to understanding the function of CUL3 as well as Eg5 in mitosis. Further characterization of the interaction between CUL3 and Eg5 demonstrated that this interaction is present only in the presence of depolymerized microtubules. Our results culminate to the following conclusions: CUL3 is required for cells to properly undergo mitosis, especially the correct formation of spindle apparatus. CUL3 interacts with the spindle assembly factor Eg5 in the presence of depolymerized microtubules, specifically during mitosis. We suspect that CUL3-Eg5 interaction is important for mitosis and in particular for bipolar spindle formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-288. doi:1538-7445.AM2012-LB-288