Overview
Our laboratory applies experimental and computational approaches to the analysis of chromosome segregation, genomic stability and programmed cell death in yeast, mice and human cells. Our overall approach is to link systems-wide models of cellular processes with detailed mechanistic information on the activities of individual proteins. We are particularly interested in the microtubule-based machines that power chromosome movement during cell division, the mitotic checkpoints that ensure the accuracy of chromosome segregation, and the signal transduction systems that control programmed cell death. We believe that our research will have long-term practical benefits in revealing connections between chromosome instability (CIN) and cancer, in elucidating the mechanisms of action of anti-microtubule chemotherapeutics, and in the generation of predictive models of cell signaling that can be used for drug discovery.

Research Summary
Students, postdocs and research scientists in our laboratory are actively involved in the development of technologies for systematic experimental analysis and in the application of these technologies to biological problems. In collaboration with engineering and computer science groups, we are working on micro-fabricated biosensors that can measure the levels, activities and states of modification of proteins in living cells and cell extracts. We are also a founding laboratory in the Open Microscopy Environment, a large-scale programming project that seeks to develop image informatics tools for quantitative analysis of biological data.

Analysis of budding yeast kinetochores in vitro: Kinetochores are multi-protein complexes that assemble on centromeric DNA and mediate the attachment of chromosomes to microtubules. Kinetochores have the remarkably ability to couple the energy of microtubule polymerization and depolymerization into the forces required for chromosome movement. It is essential that each sister chromatid attach to microtubules at one and only one site, and kinetochore formation is therefore tightly controlled. When assembly proceeds correctly, pairs of sisters carry two kinetochores, each of which captures microtubules emanating from a different pole. This generates a state known as bipolar attachment. Microtubule-attached chromatids undergo a complex series of oscillations back and forth along the spindle axis. Once all chromatids are correctly aligned, the connections between sisters are severed and the poleward movement of chromatids begins.

Our laboratory is studying kinetochores in yeast and human cells by addressing the following specific questions:

(i) How is kinetochore assembly regulated to satisfy the ‘one and only one’ rule?
(ii) What is the overall architecture of the kinetochore and what are the functions of individual proteins?
(iii) How is force generated and chromosome movement regulated?

One of our first aims is to generate an overall architectural model of the yeast kinetochore using cell-biological assays. Selected kinetochore proteins are also being subjected to high resolution crystallographic analysis in collaboration with Stephen Harrison’s lab at Harvard Medical School. Finally, we are working to map the functional properties of kinetochores onto our architectural model. Using high resolution imaging and machine-vision algorithms (developed in collaboration with Gaudenz Danuser’s lab at the Scripps Research Institute) we can track the movement of kinetochores in wild-type and mutant yeast with very high precision. Information on chromosome trajectories is then used to calculate accelerations and uncover the mechanistic basis of force generation.

Checkpoint controls in animal cells: Accurate chromosome segregation at mitosis is ensured both by the intrinsic fidelity of the mitotic machinery and by the operation of checkpoints that monitor chromosome-microtubule attachment. When unattached chromosomes are present, anaphase is delayed, increasing the chance that chromosome-microtubule capture can be completed successfully. Checkpoint signaling is mediated by kinetochores and by a transduction systems comprising Mad and Bub proteins.

Our laboratory studies the mitotic checkpoint using a combination of biochemical, genetic and microscope-based assays. Specifically, we are asking:

(i) How is bivalent attachment sensed by the mitotic checkpoint?
(ii) What goes wrong in tumor cells to cause chromosome instability?

To address these questions, RNAi-mediated gene inactivation and live cell microscopy are being combined to determine how the checkpoint signal is generated and propagated in human cells. In mice, both conventional and conditional knockouts are being employed to study the effects on checkpoint inactivation on chromosome segregation and tumor promotion in vivo.

Recent experiments in tissue culture cells have revealed that the spindle assembly checkpoint plays a role not only in detecting the presence of unaligned chromosomes, but also in controlling the overall timing of mitosis. This timing function requires the Mad2 and BubR1 checkpoint proteins, but, unlike the unaligned chromosome checkpoint, does not require functional kinetochores. The dramatic acceleration in mitosis that accompanies Mad2 deletion helps to explain why Mad2 is an essential gene in higher eukaryotes. However, we have recently discovered that the lethality of a mouse Mad2 deletion can be suppressed by the simultaneous elimination of the p53 tumor suppresser gene. Mad2-p53 double knockout murine cells are viable (although animals are not) and exhibit a very high degree of genomic instability. These data suggest an important role for p53-mediated apoptosis in the death of animal cells lacking checkpoint controls during mitosis.

Systems Biology of Apoptosis: It has become increasingly clear that understanding signal transduction requires the development of models that combine an understanding of the interactions among large sets of molecules with precise mechanistic information above individual proteins. These models will be numerical, but it is essential that they be formulated on the basis of experimental data. In addition, the models must be subjected to rigorous experimental verification. Thus, systems biology must focus equally on computation and experimentation.

We are part of a multi-lab MIT consortium (CSBi) whose goal is to develop the instrumentation and methods for the parallel interrogation and analysis of apoptosis in human cells. Data from these methods is then analyzed within one of several computational frameworks based on ordinary differential equations (ODEs), Bayesian networks and statistical data mining. Early successes in this project include the development (in collaboration with Doug Lauffenburger’s group at MIT) of an experimentally-based 400 equation ODE model that can capture cell-type specific variation in the generation of survival signaling emanating from the EGF receptor.

Selected Publications
Swedlow JR, Goldberg I, Brauner E, Sorger PK. Informatics and quantitative analysis in biological imaging. Science 300:100-102 (2003).

He, X., Rines, D.R., Espelin, C.W, and Sorger, P.K. Molecular analysis of kinetochore-microtubule attachment in budding yeast. Cell 106:195-206 (2001).

He, X., Asthana, S., Sorger, P.K. Transient Sister Chromatid Separation and Elastic Deformation of Chromosomes During Mitosis in Budding Yeast. Cell 102:21-33 (2000).

Dobles, M., Liberal, V., Scott, M.L., Benezra, R. and Sorger, P.K. Apoptosis and Chromosome Mis-segregation in Mice Lacking the Mitotic Checkpoint Protein Mad2. Cell 101:635-645 (2000).

Kaplan, K.B., Hyman, A.A. and Sorger, P.K. Regulating the yeast kinetochore by p23Skp1-dependent phosphorylation and ubiquitin-mediated degradation Cell 91:491-500 (1997).