Quantitative analysis of the receptor-induced apoptotic decision network

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.Includes bibliographical references (p. 157-169).Cells use a complex web of protein signaling pathways to interpret extracellular cues and decide and execute cell fates such as survival, apoptosis, differentiation, and proliferation. Cell decisions can be triggered by subtle, transient signals that are context specific, making them hard to study by conventional experimental methods. In this thesis, we use a systems approach combining quantitative experiments with computational modeling and analysis to understand the regulation of the survival-vs-death decision. A second goal of this thesis was to develop modeling and analysis methods that enable study of signals that are transient or at intermediate activation levels. We addressed the challenge of balancing mechanistic detail and ease of interpretation in modeling by adapting fuzzy logic to analyze a previously published experimental dataset characterizing the dynamic behavior of kinase pathways governing apoptosis in human colon carcinoma cells. Simulations of our fuzzy logic model recapitulated most features of the data and generated several predictions involving pathway crosstalk and regulation. Fuzzy logic models are flexible, able to incorporate qualitative and noisy data, and powerful enough to generate not only quantitative predictions but also biological insights concerning operation of signaling networks. To study transient signals in differential-equation based models, we employed direct Lyapunov exponents (DLEs) to identify phase-space domains of high sensitivity to initial conditions. These domains delineate regions exhibiting qualitatively different transient activities that would be indistinguishable using steady-state analysis but which correspond to different outcomes.(cont.) We combine DLE analysis of a physicochemical model of receptor-mediated apoptosis with single cell data obtained by flow cytometry and FRET-based reporters in live-cell microscopy to classify conditions that alter the usage of two apoptosis pathways (Type I/II apoptosis). While it is generally thought that the control point for Type I/II occurs at the level of initiator caspase activation, we find that Type II cells can be converted to Type I by removal of XIAP, a regulator of effector caspases. Our study suggests that the classification of cells as Type I or II obscures a third variable category of cells that are highly sensitive to changes in the concentrations of key apoptotic network proteins.by Bree Beardsley Aldridge.Ph.D

Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria

Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo—protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development