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.
Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600–630) and two in the far red channel (633/641–680), supplemented by a single further redshifted fluorochrome (670/701–750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.
Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were depleted with 775 nm. To the best of our knowledge, these are the first published five color STED images. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.
Eukaryotic cells decide in late G1 whether to commit to another round of genome duplication and division. This point of irreversible cell cycle commitment is a molecular switch termed “Restriction Point” in mammals and “Start” in budding yeast. At Start, yeast cells integrate multiple signals such as pheromones, osmolarity, and nutrients. If sufficient nutrients are lacking, cells will not pass Start. However, how the cells respond to nutrient depletion after they have made the Start decision, remains poorly understood.Here, we analyze by live cell imaging how post-Start yeast cells respond to nutrient depletion. We monitor fluorescently labelled 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 back into the nucleus. This occurs when cells are faced with starvation up to 20 minutes after Start. In these cells, the positive feedback loop is interrupted, Whi5 re-binds DNA, and CDK activation occurs a second time once nutrients are replenished. Cells which re-import Whi5 also become sensitive to mating pheromone again, and thus behave like pre-Start cells. In summary, we show that upon starvation the commitment decision at Start can be reversed. We therefore propose that in yeast, as has been suggested for mammalian cells, cell cycle commitment is a multi-step process, where irreversibility in face of nutrient signaling is only reached approximately 20 minutes after CDK activation at Start.
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