19The coordination of metabolism and growth with cell division is crucial for proliferation. While it 20 has long been known that cell metabolism regulates the cell division cycle, it is becoming 21 increasingly clear that the cell division cycle also regulates metabolism. In budding yeast, we 22 previously showed that over half of all measured metabolites change concentration through the 23 cell cycle indicating that metabolic fluxes are extensively regulated during cell cycle progression. 24 However, how this regulation is achieved still remains poorly understood. Since both the cell cycle 25 and metabolism are regulated to a large extent by protein phosphorylation, we here decided to 26 measure the phosphoproteome through the budding yeast cell cycle. Specifically, we chose a cell 27 cycle synchronisation strategy that avoids stress and nutrient-related perturbations of metabolism, 28 and we grew the yeast on ethanol minimal medium to force cells to utilize their full biosynthetic 29 repertoire. Using a tandem-mass-tagging approach, we found over 200 sites on metabolic enzymes 30 and transporters to be phospho-regulated. These sites were distributed among many pathways 31 including carbohydrate catabolism, lipid metabolism and amino acid synthesis and therefore likely 32 contribute to changing metabolic fluxes through the cell cycle. Among all one thousand sites 33 whose phosphorylation increases through the cell cycle, the CDK consensus motif and an arginine-34 directed motif were highly enriched. This arginine-directed R-R-x-S motif is associated with 35 protein-kinase A, which regulates metabolism and promotes growth. Finally, we also found over 36 one thousand sites that are dephosphorylated through the G1/S transition. We speculate that the 37 phosphatase Glc7/ PP1, known to regulate both the cell cycle and carbon metabolism, may play 38 an important role because its regulatory subunits are phospho-regulated in our data. In summary, 39 our results identify extensive cell cycle dependent phosphorylation and dephosphorylation of 40 metabolic enzymes and suggest multiple mechanisms through which the cell division cycle 41 regulates metabolic signalling pathways to temporally coordinate biosynthesis with distinct phases 42 of the cell division cycle.43 45division cycle, which ensures that DNA and other crucial cellular components are duplicated and 46 divided between two daughter cells. In budding yeast, it was viewed that cell metabolism and 47 growth proceed largely independently of the cell cycle. This assumption comes from the 48 observation that mutants arrested in distinct phases of the cell cycle continued to grow and became 49 extremely large and irregularly shaped (Hartwell et al., 1974;Johnston et al., 1977; Pringle and 50 Hartwell, 1981). This showed clearly that a cell cycle arrest does not stop metabolism and mass 51 accumulation, which led to the text book model that in budding yeast growth controls division, but 52 not vice versa (Morgan, 2007). 53While the hierarchy of metaboli...

Live-cell microscopy is a powerful tool that can reveal cellular behavior as well as the underlying molecular processes. A key advantage of microscopy is that by visualizing biological processes, it can provide direct insights. Nevertheless, live-cell imaging can be technically challenging and prone to artifacts. For a successful experiment, many careful decisions are required at all steps from hardware selection to downstream image analysis. Facing these questions can be particularly intimidating due to the requirement for expertise in multiple disciplines, ranging from optics, biophysics, and programming to cell biology. In this review, we aim to summarize the key points that need to be considered when setting up and analyzing a live-cell imaging experiment. While we put a particular focus on yeast, many of the concepts discussed are applicable also to other organisms. In addition, we discuss reporting and data sharing strategies that we think are critical to improve reproducibility in the field.

Metabolism and the Cell Cycle cell cycle dependent phosphorylation and dephosphorylation of metabolic enzymes and suggest multiple mechanisms through which the cell division cycle regulates metabolic signaling pathways to temporally coordinate biosynthesis with distinct phases of the cell division cycle.

Eukaryotic cells decide in late G1 phase of the cell cycle whether to commit to another round of division. This point of cell cycle commitment is termed “Restriction Point” in mammals and “Start” in the budding yeast Saccharomyces cerevisiae. At Start, yeast cells integrate multiple signals such as pheromones and nutrients, and will not pass Start if nutrients are lacking. However, how cells respond to nutrient depletion after the Start decision remains poorly understood. Here, we analyze how post‐Start cells respond to nutrient depletion, by monitoring Whi5, the cell cycle inhibitor whose export from the nucleus determines Start. Surprisingly, we find that cells that have passed Start can re‐import Whi5 into the nucleus. In these cells, the positive feedback loop activating G1/S transcription is interrupted, and the Whi5 repressor re‐binds DNA. Cells which re‐import Whi5 become again sensitive to mating pheromone, like pre‐Start cells, and CDK activation can occur a second time upon replenishment of nutrients. These results demonstrate that upon starvation, the commitment decision at Start can be reversed. We therefore propose that cell cycle commitment in yeast is a multi‐step process, similar to what has been suggested for mammalian cells.