Identification of a Potential Mitotic Function for the Mammalian Nup50
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Date
2011-06
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The Ohio State University
Abstract
Mitosis is a conserved process in which the genetic material, DNA, is equally segregated between two daughter cells. DNA is contained in the nucleus of the eukaryotic cell and surrounded by the nuclear envelope. Multi protein complexes known as the Nuclear Pore Complexes (NPCs) embed within the nuclear envelope and regulate the transport of molecules in and out of the nucleus. Surprisingly, in Aspergillus nidulans, the model system used in my study, a nuclear pore complex protein Nup2 undergoes a unique translocation to chromatin during mitosis and is essential for proper mitotic progression. Interestingly, the Nup2 homolog in higher eukaryotes, Nup50, undergoes the same translocation. Therefore, the purpose of this study is to test whether Nup50 can translocate onto chromatin in Aspergillus nidulans and complement the mitotic function of Nup2. In order to test this hypothesis, the Nup50 gene was integrated into A. nidulans using homologous recombination. Four way fusion PCR was used to generate a DNA cassette that contains the Nup50 gene fused to EGFP2 marker and its expression under control of the inducible promoter alcA. Once Nup50 was introduced into A. nidulans, Nup2 was deleted in the background. So far, we have discovered that Nup50 is present in the nucleus at interphase and disperses throughout the cell during mitosis in the absence or presence of the Aspergillus nidulans Nup2. Results showed that Nup50 cannot complement Nup2’s function perhaps because it is not located at either the NPCs of mitotic chromatin. Furthermore, mmNup50 is a much smaller protein than Nup2 and it lacks a specific domain which is present in Nup2, termed domain 4. Domain 4 has been shown to be sufficient and necessary for Nup2’s location to the pore and DNA. This suggests that introducing domain 4 into mmNup50 might tether it to the pore at interphase and mitotic chromatin. We plan to create a version of Nup50 that carries domain 4 and replace the endogenous An-Nup2 with this version. We can then ask whether this version of Nup50 can complement Nup2’s function. This study may provide evidence that the novel essential mitotic function of the A. nidulans Nup2 is conserved in humans. Knowledge gained from these experiments might help us identify a potentially novel essential function of the mammalian Nucleoporin 50 in mitosis, which is important to understanding the initiation and progression of human diseases that can arise from defective mitosis such as cancer.
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Keywords
mitosis, nup2, nup50, NPC