Mitogen-activated protein kinase (MAPK) dynamics determine cell fate in the yeast mating response

Abstract

In the yeast Saccharomyces cerevisiae, the exposure to mating pheromone activates a prototypic mitogen-activated protein kinase (MAPK) cascade and triggers a dose-dependent differentiation response. Whereas a high pheromone dose induces growth arrest and formation of a shmoo-like morphology in yeast cells, lower pheromone doses elicit elongated cell growth. Previous population-level analysis has revealed that the MAPK Fus3 plays an important role in mediating this differentiation switch. To further investigate how Fus3 controls the fate decision process at the single-cell level, we developed a specific translocation-based reporter for monitoring Fus3 activity in individual live cells. Using this reporter, we observed strikingly different dynamic patterns of Fus3 activation in single cells differentiated into distinct fates. Cells committed to growth arrest and shmoo formation exhibited sustained Fus3 activation. In contrast, most cells undergoing elongated growth showed either a delayed gradual increase or pulsatile dynamics of Fus3 activity. Furthermore, we found that chemically perturbing Fus3 dynamics with a specific inhibitor could effectively redirect the mating differentiation, confirming the causative role of Fus3 dynamics in driving cell fate decisions. MAPKs mediate proliferation and differentiation signals in mammals and are therapeutic targets in many cancers. Our results highlight the importance of MAPK dynamics in regulating single-cell responses and open up the possibility that MAPK signaling dynamics could be a pharmacological target in therapeutic interventions.

Keywords: cell fate decision; cell signaling; differentiation; imaging; mitogen-activated protein kinase (MAPK); pheromone; single-cell analysis; yeast mating response.

Figure 1.

Development of a KTR for Fus3 activity. A, top panel, diagram of the MAPK signaling pathway that mediates mating differentiation in yeast. Bottom panel, representative images of cells undergoing elongated growth and shmoo formation. B, schematic of the KTR for Fus3 developed in this study. Green regions, NLS; blue regions, NES. C, representative time-lapse images showing the reporter translocation in response to pheromone stimulation. D, average time traces of reporter translocation response to a 30-min pulse of 1 μm pheromone treatment. The reporter response was quantified as the cytoplasmic over nuclear fluorescence intensities (C/N ratio) and was normalized by the basal level. Means and S.E. are shown for WT (blue curve), kss1Δ (red curve), and fus3Δ (green curve). E, average time trace of reporter response in the FUS3^as, KSS1 strain (blue curve). Cells were exposed to a constant 1 μm pheromone treatment, and 10 μm 1-NM-PP1 was added 30 min after pheromone stimulation. The response from cells without pheromone exposure was shown as a control (gray curve).

Figure 2.

Cells with different fates exhibit distinct dynamics of Fus3 activation. A and B, time traces of Fus3 activation in individual cells with different fates. Top panels, time-lapse phase images for representative cells undergoing elongated growth (A) or shmoo formation (B). Bottom panels, time traces of Fus3 activation in representative single cells undergoing elongated growth (A) or shmoo formation (B). C and D, single-cell color map trajectories of Fus3 activation for all of the cells that underwent elongated growth in response to 0.25 μm pheromone treatment (C) or all of the cells committed to growth arrest and shmoo formation in response to 1 μm pheromone treatment (D). Each row represents the time trace of a single cell. Color represents the normalized C/N ratio, as indicated in the color bar. E, boxplot showing the time-dependent distributions of gene expression responses in cells undergoing elongated growth (blue) and shmoo formation (red). Data are from the same cells in C and D. In the plot, the bottom and top of the box are first (the 25th percentile of the data, q1) and third quartiles (the 75th percentile of the data, q3); the band inside the box is the median; the dashed curve is the mean; the whiskers cover the range between q1 − 1.5 × (q3-q1) and q3 + 1.5 × (q3-q1).

Figure 3.

Chemical perturbation of Fus3 dynamics redirects cell fate decision. A, average time traces of Fus3 activity in response to 1 μm pheromone treatment (red curve) or 1 μm pheromone treatment + 0.5 μm inhibitor treatment (blue curve). Top panel, schematic showing the perturbation pattern: 0.5 μm 1-NM-PP1 was added after 10 min of 1 μm pheromone treatment. Means and S.E. are shown for both the control and perturbation conditions. B, bar graphs showing fractions of cells exhibiting round, elongated, or shmoo morphology upon 1 μm pheromone treatment or 1 μm pheromone treatment + 0.5 μm inhibitor treatment. Data are from the same cells in A. C and D, lower panels, single-cell color map trajectories of Fus3 activity for all of the cells that underwent elongated growth in response to 1 μm pheromone treatment + 0.5 μm inhibitor treatment (C) or all of the cells committed to growth arrest and shmoo formation in response to 1 μm pheromone treatment (D). The color scheme is identical to that in Fig. 2, C and D. Top panels, time-lapse phase images for representative cells under the control or the perturbation condition.