Understanding Cell Division in Normal and Cancer Cells
The goal of our research program is to understand how cell division is regulated, both in normal cells and in cancer cells. We are especially interested in the role of ubiquitin-dependent proteolysis in regulating progression through mitosis. To approach these problems, weintegrate biochemical, cell biological, and chemical approaches.
Ubiquitin-Dependent Degradation During Mitosis
Several steps in cell division are regulated by ubiquitin-dependent proteolysis. The transition from metaphase to anaphase is initiated by the degradation of proteins that regulate chromosome cohesion, and the exit from mitosis is dependent on the degradation of mitotic cyclins. These proteins are marked for degradation by a multisubunit ubiqitin-protein ligase called the Anaphase Promoting Complex or Cyclosome (APC/C). Through high-throughput screens performed in Xenopus extracts (Verma et al., 2004), our laboratory has identified chemicals that inhibit ubiquitin-dependent degradation by binding the ubiquitin chain (Verma et al., 2004), and more recently compounds that inhibit APC activation by perturbing activator protein binding (Zeng et al., 2010). We are using these small molecules to study the mechanism of APC/C-dependent ubiquitination, and the role of the APC/C in cell division.
We are also interested in the question of how ubiquitinated proteins are targeted for degradation by the proteasome. In collaboration with the Gygi and Finley labs at HMS, we have reconstituted proteasome-dependent degradation of ubiquitinated cyclin B1 from purified components. We have used quantitative mass spectrometry to determine how the APC/C ubiquitinates its substrates (Kirkpatrick et al., 2006), and we are currently working to understand the minimal ubiquitin signal that is essential for rapid degradation by the proteasome. In collaboration with the Finley lab, we have characterized the role of the proteasome-associated deubiquitinating enzyme Ubp6 (Hanna et al., 2006) and recently its human ortholog, Usp14 (Lee et al., 2010). We have recently identified a selective small molecule inhibitor of Usp14 that can enhance the cell's ability to eliminate potentially neurotoxic or damaged proteins (Lee et al., 2010). These findings may provide a new method for treatment of diseases associated with protein misfolding, such as neurodegenerative diseases.
Protein degradation regulates exit from mitosis. Anaphase is initiated when the securin proteins are targeted for ubiquitination and subsequent degradation by the Anaphase-Promoting Complex/Cyclosome. The cell exits mitosis and returns to interphase when cyclins are destroyed by a similar mechanism. We are using small molecule inhibitors of the ubiquitination reaction to study this pathway in mammalian cells.
Causes and Consequences of Chromosome Missegregation
Many cancers have defects in the mechanisms that ensure the proper segregation of chromomes to daughter cells. As a result, cancer cells gain and lose chromomes as they divide. Our goal is to understand the molecular basis of these defects, and to determine whether defects in chromosome segregation can be exploited for the development of new cancer therapies. We have developed long-term time-lapse imaging as a tool for understanding the source of chromsome segregation errors in cells, and the consequences of segregation errors. We have found that spontaneous chromosome nondisjunction is coupled to cytokinesis completion in human cell lines (Shi and King, 2005). Our data suggest that tetraploid cells may arise quite frequently, thus serving as a potential intermediate for the ultimate generation of aneuploid cells. We are currently working to understand the cause(s) of spontaneous segregation errors, and how these errors relate to the regulation of cytokinesis. For reviews, see Normand and King, 2010 and King, 2008.
Watching Cells Divide: A metaphase cell expressing a cyclin B-GFP fusion protein. This version of the protein binds to condensed chromosomes. Time-lapse microscopy of cells is used in the lab to study the effect of small-molecule inhibitors of cell division. Image courtesy of Jon Hoyt.
Large Scale Screening
Incollaborationwith the Institute of Chemistry and Cell Biologyat HMS, our laboratory is continuing to develop screens for novel small molecules or genes that regulate mitosis and ubiquitin-dependent proteolysis. We have developed methods for screening large chemical libraries in Xenopus cell cycle extracts (Verma et al., 2004). We have also used high-throughput automated imaging to identify small molecules or siRNAs that perturb mitosis in human cells. These screens have identified several novel regulators of mitosis whose mechanism of action we are continuing to explore.
A sample image from a high-throughput luciferase-based assay. The assay plate was designed in our lab and contains 1536 wells that hold 2 microliters each. This system has allowed us to screen libraries of hundreds of thousands of compounds to identify small molecules that interfere with cell division.
Automated Long-Term Imaging
To study the regulation of cell division, we have employed long-term time-lapse imaging. We routinely image cells for 2-6 day periods in 96 well plates using a variety of different fluorescent reporter proteins. This method enables us to understand how small molecules or knock-down of different genes affects timing of different events in the cell cycle. In collaboration with Steve Wong's group (Methodist Hospital, Houston) we are developing software for automated analysis of the movies that are generated by this approach (Wang et al., 2008).