Metabolic rhythms occur in different cells and compartments within organisms. The origins and impact of these rhythms on other biological oscillators (e.g. cell cycle, circadian clocks) is only starting to be understood. The applicant?s long-term goal is to understand the mechanisms, function, and interaction of metabolic rhythms and the cell cycle in budding yeast, a model eukaryote. The yeast metabolic cycle (YMC) is a synchronous metabolic rhythm that occurs in a chemostat. The population synchrony arises from YMC-to-YMC coupling between cells via secreted metabolites. The YMC within each cell also interacts with the cell division cycle (CDC) to coordinate the events of carbon catabolism and cell cycle entry. These two oscillators have different periods, yet remain coordinated such that a fraction of the population commits to the CDC each YMC. Inference of YMC-CDC dynamics has been challenging because metabolic and cell cycle events are often measured and averaged across a heterogeneous population, which masks the dynamics that occur in a single cell. The objective of this proposal is to obtain new insights into these intracellular oscillators by measuring and perturbing the YMC and CDC in single cells. The central hypothesis is that the YMC and CDC can oscillate independently of one another but are normally coordinated in yeast through a reinforcing feedback loop (i.e., entry into a carbon catabolic state triggers the cell cycle, and, reciprocally, initiating the cell cycle triggers entry into a carbon catabolic state). The applicants will generate data that address the central hypothesis and its alternative with three specific aims: (1) Develop fluorescent reporter assays to measure population snapshots of metabolic and cell cycle states in single cells taken from a cycling chemostat; (2) Perturb the reinforcing feedback loop to disrupt the synchronization of YMC and CDC events in a cycling chemostat; and (3) Measure and perturb YMC-CDC dynamics outside the chemostat using timelapse fluorescence microscopy with microfluidics.
Aim 1 will elucidate the timing and coordination of metabolic and cell cycle events in a cycling chemostat across different growth conditions.
Aim 2 will directly test the reinforcing feedback loop that coordinates these oscillators in a cycling chemostat.
Aim 3 will measure the extent to which metabolic rhythms occur in the absence of cell-to-cell communication via secreted metabolites and whether they remain coordinated with cell cycle events as seen in the chemostat. The observation that carbon catabolism and cell cycle entry remain coordinated in single cells across diverse growth conditions would strongly support the central hypotheses. This work is innovative because it combines single-cell technology and molecular genetics to address an unsolved problem in yeast with broad relevance to metabolic rhythms and cell cycle in other eukaryotes. This proposal is significant because it elucidates new mechanisms and regulatory principles of how intracellular oscillators with different frequencies can interact and remain functional.

Public Health Relevance

The proposed research is relevant to public health because circadian clocks, metabolic rhythms, and the cell division cycle are intricately linked, and disrupting their interactions can lead to disease progression. A deeper understanding of how these eukaryotic oscillators have evolved to interact with each other may suggest novel therapeutic approaches to treat human diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Melillo, Amanda A
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North Carolina State University Raleigh
Veterinary Sciences
Schools of Veterinary Medicine
United States
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