Project Summary Age- and function-based positioning of mitochondria plays a critical and conserved role in cellular differentiation and aging from yeast to humans. Evidence suggests that mitochondrial positioning mechanisms selectively transport or retain specific mitochondria based on their fitness, resulting in physiological asymmetry between mother and daughter cells. However, a major conceptual challenge is that mitochondria form connected networks. They experience constant remodeling by mitochondrial fission and fusion, while their size continuously expands during cell growth. The continuous mitochondrial content mixing should eliminate any differences between mitochondria, making it difficult to explain how some mitochondria can be fitter than others. In this proposal, we unite and integrate complementary expertise in mitochondrial biology, quantitative cell physiology, and in silico modeling of organelle dynamics to mechanistically investigate the precise sequence of subcellular events that lead to asymmetric mitochondrial segregation, first during the cell cycle and ultimately to replicative aging. We will implement powerful molecular and single-cell techniques in combination with in silico modeling approaches to control, track, and model individual mitochondria and their dynamics, topology, and function as cells age. The true power and innovation of the proposed research is the tight integration of approaches guided by novel mechanistic hypotheses of cellular aging. In Aim 1, we will test our model for the molecular basis of fitness recognition, which centers on the role of cardiolipin (CL), a lipid linked to myriad mitochondrial functions. We hypothesize that CL both enhances the import of mitochondrial components and is selectively recognized by the mitochondrial transport and tethering proteins, providing a novel testable mechanism for the inheritance of the fittest. We will test whether the tenets of our model enable the emergence of asymmetry in mitochondrial content and function during the cell cycle and, in Aim 2, investigate the impact of that asymmetry on replicative aging. In addition, we will consider the role of other organelles such as the vacuole. We will utilize the yeast mother machine to reveal a hierarchy of age-related changes in mitochondria and vacuoles. The causal relationship between cellular aging and age-dependent changes in mitochondria and the vacuole will be determined. Our studies in yeast will uncover fundamental mechanisms used by cells to establish functional asymmetry within mitochondrial networks. Therefore, this work has the potential to lead to novel therapeutic strategies to slow or reverse aging in humans by manipulating the mitochondrial drivers of aging at the cellular level.