Research
We are a basic cancer research lab with focus on the cell biology of mitosis. The cancer model we focus on is breast cancer. Mitosis is not simply a cell proliferation process. The connections with cell signaling pathways make mitosis an effective way to generate different varieties of daughter cells through one cell division.
We have long investigated both subcellular structures (centromeres, kinetochores, and centrosomes) and regulatory mechanisms (mitotic checkpoint) involved in the prevention of aneuploidy and chromosomal instability. More recently we have also developed interest in cell division symmetry control and mitochondria inheritance. We believe these work will help clarify how cancer cells gain genetic, epigenetic and functional heterogeneity.
Current Projects:
1. Activation of the spindle assembly checkpoint (SAC): MAD2 O-C conversion, and the 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, a cellular hallmark 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 in human cells. Biochemical purification and reconstitution, together with analyses using mitotic cell lysates and live cell imaging, are major research approaches. How C-MAD2 is produced, how MCC is assembled and how MCC inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) are being investigated.
2. 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 by disassembling MCC [link]. KIAA1377/Cep126 is a novel centrosome protein that also interacts with p31comet. Despite the many wonderful structural models of the MCC and APC/C complexes, how to coordinate the activation and silencing to maintain both sensitivity and robustness of the checkpoint still needs a lot of work.
3. The centromere/kinetochore complex in human cells
Are we done with cataloging the human centromere/kinetochore proteins? 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 prevents chromosome missegregation also originate from the complex. We have compiled a comprehensive list of over 200 centromere/kinetochore proteins in human cells. 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.
4. Functional characterization of cancer "signature" genes: Surprisingly the biological functions are still unclear for many genes listed in common cancer gene signatures. Some are co-expressed with core centromere/kinetochore components. We focus on chromosomal instability (CIN) signature genes in breast cancers.
5. Aneugen, Aneuploidy and Breast Cancer: Diazepam and mitotic cell death; Aneuploidy tolerating mutations.
Major Scientific Achievements of the Lab:
1. Searching for novel mitosis regulators: Compiled a comprehensive list of >200 experimentally validated centromere/kinetochore proteins in human cells; Identified novel mitosis regulators based on data mining: TRIP13, KIAA1377/Cep126 etc.
2. Activation of the mitotic checkpoint: In establishing the connection between the signal transducer and the effector, we first reported that intracellular C-MAD2 concentration is the major determinant for MCC assembly; first reported and characterized direct interaction between BUBR1 and C-MAD2; demonstrated that a fully assembled MCC is critical in inhibiting mitotic APC/C; provided evidence that MPS1 kinase is required for C-MAD2 production; showed that MPS1 phosphorylation of MAD1 and less appreciated MAD1 N-terminal and C-terminal domains are essential for C-MAD2 production; ...
3. Silencing of the mitotic checkpoint: By inquiring the energy requirement during mitotic checkpoint silencing, we first identified and characterized TRIP13 AAA-ATPase as a mitotic checkpoint silencing protein; first proposed the functional importance of C to O-MAD2 conversion in silencing the mitotic checkpoint and the role of TRIP13 in the conversion; ...
Research Resources: lab safety, research ethics and compliance, Undergraduate Research, journals, databases, scientific tools...
The research techniques a trainee can expect to learn:
Molecular cloning, Gateway Technology, mammalian and insect cell culture, DNA transfection, siRNA and shRNA, CRISPR, 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, 3D cell culture, xenograft mouse, transgenic mouse, etc....