Autonomous Metabolic Oscillations Robustly Gate the Early and Late Cell Cycle.

Eukaryotic cell division is known to be controlled by the cyclin/cyclin dependent kinase (CDK) machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic triggers for cell-cycle initiation and progression. Using microfluidics, cell-cycle reporters, and single-cell metabolite measurements, we found that metabolism of budding yeast is a CDK-independent oscillator that oscillates across different growth conditions, both in synchrony with and also in the absence of the cell cycle. Using environmental perturbations and dynamic single-protein depletion experiments, we found that the metabolic oscillator and the cell cycle form a system of coupled oscillators, with the metabolic oscillator separately gating and maintaining synchrony with the early and late cell cycle. Establishing metabolism as a dynamic component within the cell-cycle network opens new avenues for cell-cycle research and therapeutic interventions for proliferative disorders.

Temporal segregation of biosynthetic processes is responsible for metabolic oscillations during the budding yeast cell cycle.

Many cell biological and biochemical mechanisms controlling the fundamental process of eukaryotic cell division have been identified; however, the temporal dynamics of biosynthetic processes during the cell division cycle are still elusive. Here, we show that key biosynthetic processes are temporally segregated along the cell cycle. Using budding yeast as a model and single-cell methods to dynamically measure metabolic activity, we observe two peaks in protein synthesis, in the G1 and S/G2/M phase, whereas lipid and polysaccharide synthesis peaks only once, during the S/G2/M phase. Integrating the inferred biosynthetic rates into a thermodynamic-stoichiometric metabolic model, we find that this temporal segregation in biosynthetic processes causes flux changes in primary metabolism, with an acceleration of glucose-uptake flux in G1 and phase-shifted oscillations of oxygen and carbon dioxide exchanges. Through experimental validation of the model predictions, we demonstrate that primary metabolism oscillates with cell-cycle periodicity to satisfy the changing demands of biosynthetic processes exhibiting unexpected dynamics during the cell cycle.

Proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization.

Microtubules are polarized intracellular polymers that play key roles in the cell, including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule-organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome; however, most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays, which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based, proximity-labeling approach TurboID for use in C. elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B/MACF1 and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease protein WDR62. VAB-10B and WDR-62 do not associate with the centrosome and instead specifically regulate non-centrosomal microtubules and the apical targeting of microtubule minus-end proteins. Depletion of VAB-10B resulted in microtubule mislocalization and delayed localization of a microtubule nucleation complex É£-tubulin ring complex (γ-TuRC), while loss of WDR-62 decreased the number of dynamic microtubules and abolished γ-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, we expand the basic cell biological roles of this important disease protein. Our studies identify essential ncMTOC components and suggest a division of labor where microtubule growth and localization are distinctly regulated.

Inference of the High-Level Interaction Topology between the Metabolic and Cell-Cycle Oscillators from Single-Cell Dynamics.

Recent evidence suggests that the eukaryotic metabolism is an autonomous oscillator. Together with oscillating elements of the cyclin/CDK machinery, this oscillator might form a coupled oscillator system, from which cell-cycle control emerges. The topology of interactions between the metabolic oscillator and the elements of the cyclin/CDK machinery, however, remains unknown. Using single-cell metabolic and cell-cycle dynamics in yeast, and solving an inverse problem with a system of Kuramoto oscillators, we inferred how the metabolic oscillator interacts with the cyclin/CDK machinery. The identified and experimentally validated interaction topology shows that the early and late cell cycle are independently driven by metabolism. While in this topology, the S phase is coordinated by START. We obtained no support for a strong interaction between early and late cell cycle. The identified high-level interaction topology will guide future efforts to discover the molecular links between metabolism and the cell cycle.

Saccharomyces cerevisiae goes through distinct metabolic phases during its replicative lifespan.

A comprehensive description of the phenotypic changes during cellular aging is key towards unraveling its causal forces. Previously, we mapped age-related changes in the proteome and transcriptome (Janssens et al., 2015). Here, employing the same experimental procedure and model-based inference, we generate a comprehensive account of metabolic changes during the replicative life of Saccharomyces cerevisiae. With age, we found decreasing metabolite levels, decreasing growth and substrate uptake rates accompanied by a switch from aerobic fermentation to respiration, with glycerol and acetate production. The identified metabolic fluxes revealed an increase in redox cofactor turnover, likely to combat increased production of reactive oxygen species. The metabolic changes are possibly a result of the age-associated decrease in surface area per cell volume. With metabolism being an important factor of the cellular phenotype, this work complements our recent mapping of the transcriptomic and proteomic changes towards a holistic description of the cellular phenotype during aging.

Differential scaling between G1 protein production and cell size dynamics promotes commitment to the cell division cycle in budding yeast.

In the unicellular eukaryote Saccharomyces cerevisiae, Cln3-cyclin-dependent kinase activity enables Start, the irreversible commitment to the cell division cycle. However, the concentration of Cln3 has been paradoxically considered to remain constant during G1, due to the presumed scaling of its production rate with cell size dynamics. Measuring metabolic and biosynthetic activity during cell cycle progression in single cells, we found that cells exhibit pulses in their protein production rate. Rather than scaling with cell size dynamics, these pulses follow the intrinsic metabolic dynamics, peaking around Start. Using a viral-based bicistronic construct and targeted proteomics to measure Cln3 at the single-cell and population levels, we show that the differential scaling between protein production and cell size leads to a temporal increase in Cln3 concentration, and passage through Start. This differential scaling causes Start in both daughter and mother cells across growth conditions. Thus, uncoupling between two fundamental physiological parameters drives cell cycle commitment.

Quantitative characterization of the auxin-inducible degron: a guide for dynamic protein depletion in single yeast cells.

Perturbations are essential for the interrogation of biological systems. The auxin-inducible degron harbors great potential for dynamic protein depletion in yeast. Here, we thoroughly and quantitatively characterize the auxin-inducible degron in single yeast cells. We show that an auxin concentration of 0.25 mM is necessary for fast and uniform protein depletion between single cells, and that in mother cells proteins are depleted faster than their daughters. Although, protein recovery starts immediately after removal of auxin, it takes multiple generations before equilibrium is reached between protein synthesis and dilution, which is when the original protein levels are restored. Further, we found that blue light, used for GFP excitation, together with auxin results in growth defects, caused by the photo-destruction of auxin to its toxic derivatives, which can be avoided if indole-free auxin substitutes are used. Our work provides guidelines for the successful combination of microscopy, microfluidics and the auxin-inducible degron, offering the yeast community an unprecedented tool for dynamic perturbations on the single cell level.