Project Summary Aging can be characterized as the progressive loss of homeostasis starting at maturity and ending with senescence and death. Discoveries of the molecular mechanisms of aging can enable the development of new therapies to block age-associated disease and extend healthspan, the healthy years of life. This goal has been elusive even in the simplest model eukaryote, single-celled budding yeast. Yeast longevity has been associated with each of hundreds of genes. Such complexity suggests that yeast aging is controlled by interactions between many molecules and organelles, with any link in this network potentially subject to compromise during a given cell division. But the chain of molecular events by which homeostasis is gradually lost has been obscure to date, in part due to the reliance of the field on bulk-culture measurements in the study of the genomics of aging. The premise of the current proposal is that dissecting the breakdown of the network in many single cells — observing its many facets over time, perturbing them, and analyzing their response, is critical for a molecular understanding of the phenomenon of aging. Toward this end, we propose to analyze yeast molecular aging trajectories via comprehensive single-cell profiling of protein reporters (Aim 1), to connect lifespan extending mutations to their downstream effectors through systematic epistasis analysis (Aim 2), and to test whether perturbing critical genes at a particular point in life (just-in-time interventions) can decrease mortality rates early in life and/or extend lifespan (Aim 3). Together, these experiments will shed light on the molecular events of the breakdown of homeostasis with age in yeast, identify dynamic interventions for rejuvenation, and reveal novel aging genes and mechanisms to serve as prime candidates for testing in metazoans.