John Bachman Harvard Medical School Title: A Quantitative Synthetic Approach to Understanding the Mitochondrial Apoptotic Switch Abstract: The Bcl-2 family proteins constitute the core regulatory machinery of apoptosis, and as such their structure, function, and interactions have been the subject of intense scrutiny. However, while many individual interactions between the family members have been experimentally characterized, the systems-level properties of the Bcl-2-regulated apoptotic switch, and the precise set of interactions that confer these properties, remain obscure. In this project we attempt to develop a rigorous understanding of the steady state and dynamic properties of the Bcl-2 network by coupling an in vitro biochemical model system with quantitative measurement and mathematical modeling. We measure the pore formation kinetics of synthetic lipid vesicles or isolated mitochondria incubated with a subset of purified Bcl-2 family proteins and use these measurements to rigorously distinguish between competing mechanistic hypotheses for Bcl-2 protein function. Efficient and transparent enumeration of an array of mechanistic alternatives is facilitated by the software framework PySB, which streamlines rule-based modeling. In addition to clarifying the context-dependence of the so-called "direct" and "indirect" activation models of Bcl-2 action, this approach has the potential to identify novel aspects of Bcl-2 protein interactions that may have been overlooked by genetic approaches.