I am a PhD Candidate at Colorado State University in Fort Collins, Colorado.
Uncovering the role of a newly identified mitotic protein, BuGZ, to understand its cancer lethality.
Our lab is interested in studying the process of cell division, mitosis, and how errors in this process can lead to genetic defects and cancers. Essential to mitosis is the attachment of each sister chromatid to spindle microtubules emanating from opposite spindle poles. The attachment occurs at the region of the chromatid known as a kinetochore, which is composed of around 100 proteins. The kinetochore is the focus of our lab’s work. We recently identified a new kinetochore protein that plays an important role during cell division, called BuGZ. We are interested in this protein because we find Glioblastoma cancer cells appear to require it for survival, while normal, healthy neural cells do not. To determine if BuGZ is truly a candidate for cancer therapies in the future, we must first ascertain what exactly it is doing during cell division. We currently know that it is important for the stability of a second protein, Bub3, which has known roles in the spindle assembly checkpoint. This checkpoint is critical for faithful segregation of the DNA, as it arrests cells in mitosis until all the proper attachments to the DNA are made, upon which the cell will be permitted to divide its DNA into two new cells and exit mitosis. We aim to determine if this link between BuGZ and Bub3 is at the root of its cancer lethality, or if BuGZ has additional Bub3-independent roles in mitosis. Our research is done in human cells and we use high resolution microscopy to definitively determine the phenotype in both cancer and healthy cells depleted of BuGZ. Preliminary data shows severe chromosome alignment defects in cancer cells depleted of BuGZ, while the healthy cells depleted of BuGZ do not show as severe defects. We are working to determine the cause of the alignment defects upon loss of BuGZ. Ultimately, we hope that the information gained from our research will contribute to the understanding of the fundamental characteristics of cancer cells and their ability to survive in condition that should lead to apoptosis.
Abstract: Aneuploidy, a condition that results from unequal partitioning of chromosomes during mitosis, is a hallmark of many cancers, including those caused by human papillomaviruses (HPVs). E6 and E7 are the primary transforming proteins in HPV that drive tumor progression. In this study, we stably expressed E6 and E7 in non-cancerous RPE1 cells and analyzed the specific mitotic defects that contribute to aneuploidy in each cell line. We find that E6-expression results in multiple chromosomes associated with one or both spindle poles, causing a significant mitotic delay. In most cells, the mis-aligned chromosomes eventually migrated to the spindle equator, leading to mitotic exit, however in some cells, mitotic exit occurred in the presence of pole-associated chromosomes. We determined that this premature mitotic exit is due to defects in spindle assembly checkpoint (SAC) signaling, such that cells are unable to maintain a prolonged mitotic arrest in the presence of unaligned chromosomes. This SAC defect is in part caused by a loss of kinetochore-associated Mad2 in E6-expressing cells, which is likely due to Mad2 degradation, as proteasome inhibition rescued Mad2 levels. Our results demonstrate that E6-expressing cells exhibit previously unappreciated mitotic defects that likely contribute to HPV-mediated cancer progression.
Pub.: 26 May '17, Pinned: 30 Jun '17
Abstract: Equal chromosome segregation requires proper assembly of many proteins, including Bub3, onto kinetochores to promote kinetochore-microtubule interactions. By screening for mitotic regulators in the spindle envelope and matrix (Spemix), we identify a conserved Bub3 interacting and GLE-2-binding sequence (GLEBS) containing ZNF207 (BuGZ) that associates with spindle microtubules and regulates chromosome alignment. Using its conserved GLEBS, BuGZ directly binds and stabilizes Bub3. BuGZ also uses its microtubule-binding domain to enhance the loading of Bub3 to kinetochores that have assumed initial interactions with microtubules in prometaphase. This enhanced Bub3 loading is required for proper chromosome alignment and metaphase to anaphase progression. Interestingly, we show that microtubules are required for the highest kinetochore loading of Bub3, BubR1, and CENP-E during prometaphase. These findings suggest that BuGZ not only serves as a molecular chaperone for Bub3 but also enhances its loading onto kinetochores during prometaphase in a microtubule-dependent manner to promote chromosome alignment.
Pub.: 28 Jan '14, Pinned: 30 Jun '17
Abstract: During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora B kinase activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologs are highly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest that BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes.
Pub.: 28 Jan '14, Pinned: 30 Jun '17
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