The theme of our research is to understand how high-fidelity chromosome segregation is achieved in normal mitosis and how cell division can go awry to affect human health. We are interested in learning about both the execution machinery and the regulatory mechanisms during mitosis.
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1. The centromere/kinetochore complex in human cells: Characterization of a novel kinetochore protein TRIP13
The centromere/kinetochore complex connects chromosomes with spindle microtubules during mitosis. Not only chromosome movement is mainly regulated by proteins located at the centromere/kinetochore complex, the spindle assembly checkpoint (SAC) signals that control the fidelity of chromosome segregation also originate from the complex. We have compiled a comprehensive list of 199 centromere/kinetochore proteins in human cells. Data mining of genomic-scale co-transcription profiling and proteomic-scale protein-protein interaction (PPI) analyses have led to the discovery of TRIP13 as a novel centromere/kinetochore protein. We are searching for additional components of the centromere/kinetochore complex and are also interested in dissecting how the complex is assembled and disassembled dynamically to meet functional requirements.
2. Activation of the spindle assembly checkpoint (SAC): BUBR1:C-MAD2 interaction in MCC assembly and function
The spindle assembly checkpoint (or simply the mitotic checkpoint) monitors kinetochore-spindle connections to control the timing of chromosome segregation. Aberrant functioning of the checkpoint has been linked to aneuploidy, which is observed in >90% of solid tumors. We are studying how the Mitotic Checkpoint Complex (MCC) is assembled from BUBR1, BUB3, MAD2 and CDC20 to form a potent effector for the SAC. Biochemical purification and reconstitution, together with analyses using mitotic cell lysates and live cell imaging, are major research approaches. We have provided evidence that only C-MAD2 is incorporated into the MCC. We were also the first to report direct interaction between BUBR1 and C-MAD2, which
has recently been confirmed in the crystal structure of the S. pombe MCC (naturally lacking BUB3). How C-MAD2 is produced, how MCC is assembled and how MCC inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) are being investigated.
3. Silencing of the SAC: How C-MAD2 is converted back to O-MAD2 and how MCC is disassembled
Silencing of the SAC is essential for proper completion of cell division. The SAC silencing involves events at two venues: at attached kinetochores and in the cytoplasm. We focus on the cytoplasmic events during SAC silencing, particularly on MCC disassembly. We also study SAC silencing by examining proteins that interact with p31comet, a known protein that is dedicated to SAC silencing. TRIP13 turns out to be a p31comet interacting protein and is involved in silencing the SAC [link].
4. Characterization of two novel centrosomal proteins: KIAA1377 and DDX39
The research techniques a trainee can expect to learn:
Molecular cloning, Gateway Technology, mammalian and insect cell culture, DNA transfection, siRNA and shRNA, immunofluorescence, live cell imaging with fluorescent proteins, in vitro translation, PCR, Western blot, immunoprecipitation/co-immunoprecipitation, pull-down assay, protein chemical cross-linking, Far-Western blot, Yeast two-hybrid, recombinant protein expression in E. coli, yeast, insect cells and mammalian cells, affinity chromatography, ion exchange chromatography, size-exclusion chromatography, column chromatography using the AKTA FPLC system, in vitro binding assay, in vitro kinase assay, in vitro ubiquitylation assay, protein degradation assay using mitotic cell extracts, two-dimensional (2-D) protein electrophoresis, etc.