When yeast cells change their mind: cell cycle "Start" is reversible under starvation.

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 microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation.

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.