Novel Approaches for Studying Mitochondrial Metabolism in Mammalian Systems

Abstract

Mitochondria are metabolic organelles essential for mammalian life. Enclosed by an inner membrane that tightly regulates the entry and egress of metabolites, the mitochondrial matrix is a specialized compartment that houses numerous metabolic processes, ranging from the synthesis of proteinogenic amino acids to the production of cellular energy. Given the unique metabolite profile of mitochondria and the fact that these organelles only make up a small portion of the total cellular material, traditional approaches using whole-cell metabolomics often fail to capture the dynamics of mitochondrial metabolism. Prior attempts to isolate mitochondria for metabolite profiling have been too slow, failing to adequately preserve the native metabolite profile, or too crude, failing to achieve sufficient purity. During my graduate studies, we developed a novel workflow for rapidly immunopurifying mitochondria from mammalian cells that offers both speed and specificity. Using our methodology in conjunction with liquid chromatography and mass spectrometry (LC/MS), we interrogated mitochondrial metabolites under different states of mitochondrial respiratory chain function, revealing numerous metabolic changes that were not seen using traditional whole-cell metabolomics. In this thesis, we now expand on this work in two ways: firstly, we present a comprehensive, step-by-step protocol for using our methodology and have included new experiments and analyses that demonstrate key aspects of our workflow; secondly, we characterize and describe the generation of a knockin mouse that we believe will allow one to utilize our methodology to study mitochondrial metabolites from specific cell types in complex tissues. Taken together, this thesis expands on our prior efforts by refining and rigorously protocolizing the original workflow and by extending our work to studies in vivo.