ABSTRACT: Studies using yeast have advanced our understanding of both replicative and chronological aging, leading to the discovery of longevity genes that have homologues in higher eukaryotes. Chronological lifespan in yeast is conventionally defined as the lifespan of a non-dividing cell. To date, this parameter has only been estimated under calorically restricted (CR) conditions, mimicked by starvation. Since post-mitotic cells in higher eukaryotes are rarely calorically-restricted, we sought to develop an alternative experimental system where non-dividing yeast would age chronologically, in the presence of excess nutrients. We report here on a system wherein alginate-encapsulated yeast are packed in a pH- and temperature-controlled bioreactor, then continuously fed non-limiting substrate for extended periods of time. We present demographic, physiological and genomic evidence indicating that after ~120 hrs, immobilized cells cease dividing, remain metabolically very active and retain >95% viability for periods of 17 days. Over the same time interval, starved planktonic cells, cultured using the same media, and also controlled for temperature and pH, retained < 1 % viability in both aerobic and anaerobic cultures,. Unlike planktonic yeast, continuously-fed immobilized cells hyper-accumulate glycogen. FACS analysis of SYTOX green-stained yeast confirms that immobilized cells completely arrest within 5 days of culture, and unlike starving planktonic cells, remain free thereafter of replicative stress and are non-apoptotic. This unusual state is supported by a global gene expression profile that is stable over time, repeatable across replicate experiments, and altogether distinct from planktonic cells cultured in the presence and absence of limiting nutrients. DNA expression profiling, performed here for the very first time on immobilized cells, reveals that glycolytic genes and their trans-acting regulatory elements are upregulated, as are genes involved in remodeling the cell wall and resisting stress; by contrast, many genes that promote cell cycle progression and carry out oxidative metabolism are repressed. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously upregulated in the immobilized state, suggesting that nutrient-sensing pathways may play a role in cell viability and longevity when yeast are immobilized and placed in prolonged culture under calorically-unrestricted conditions. The cell cycle arrest in the immobilized state is mediated by RIM 15. Over the time-course of our experiments, well-fed, non-diving immobilized cells do not appear to age. Keywords: comparison of growth states and a time course of immobilized cells Nine growth conditions or time points with two to four replicates each
