Single-cell dynamics and variability of MAPK activity in a yeast differentiation pathway

Abstract

In response to pheromones, yeast cells activate a MAPK pathway to direct processes important for mating, including gene induction, cellcycle arrest, and polarized cell growth. Although a variety of assays have been able to elucidate signaling activities at multiple steps in the pathway, measurements of MAPK activity during the pheromone response have remained elusive, and our understanding of single-cell signaling behavior is incomplete. Using a yeast-optimized FRET-based mammalian Erk-activity reporter to monitor Fus3 and Kss1 activity in live yeast cells, we demonstrate that overall mating MAPK activity exhibits distinct temporal dynamics, rapid reversibility, and a graded dose dependence around the K D of the receptor, where phenotypic transitions occur. The complex dose response was found to be largely a consequence of two feedbacks involving cyclin-mediated scaffold phosphorylation and Fus3 autoregulation. Distinct cell cycle-dependent response patterns comprised a large portion of the cell-to-cell variability at each dose, constituting the major source of extrinsic noise in coupling activity to downstream gene-expression responses. Additionally, we found diverse spatial MAPK activity patterns to emerge over time in cells undergoing default, gradient, and true mating responses. Furthermore, ramping up and rapid loss of activity were closely associated with zygote formation in mating-cell pairs, supporting a role for elevated MAPK activity in successful cell fusion and morphogenic reorganization. Altogether, these findings present a detailed view of spatiotemporal MAPK activity during the pheromone response, elucidating its role in mediating complex longterm developmental fates in a unicellular differentiation system. cell signaling | MAPK dynamics | Fus3 | mating pathway | yeast

Timing of gene expression in a cell‐fate decision system

“…If both partners are able to arrest in G1, they will extend a mating projection toward each other and polarize their sensing and secretory machinery. This will lead to a local increase in pheromone concentration that will be associated with an increase in signal transduction (Appendix Fig S20; Conlon et al , ). Mating experiments performed with a mutant unable to degrade pheromone ( bar1∆ ) clearly demonstrate that p FIG1 induction is triggered by the concentration of pheromone sensed by the cells and not by cell–cell contacts.…”

Visualizing cellular heterogeneity by quantifying the dynamics of MAPK activity in live mammalian cells with synthetic fluorescent biosensors

“…The SKARS strategy provides a global measurement of kinase activity in a cell, while FRET biosensors can monitor local changes in kinase activity 54,55 . For some studies, FRET biosensors might therefore have a clear advantage by adding a spatial information for signaling activity.…”

Quantitative analysis of the yeast pheromone pathway

“…The first was the realization that Fus3, but not Kss1, is responsible for gradient tracking (Conlon, Gelin-Licht, Ganesan, Zhang, & Levchenko, 2016;Erdman & Snyder, 2001;Errede, Vered, Ford, Pena, & Elston, 2015;Hao et al, 2008;Hegemann et al, 2015; although another group reported a specific requirement for Kss1; Paliwal et al, 2007). This was surprising to us given that Fus3 and Kss1 are both activated by the same upstream protein kinases, and either MAPK can sustain mating transcription and elongated growth (Breitkreutz et al, 2001;Conlon et al, 2016;Erdman & Snyder, 2001;Errede et al, 2015;Hao et al, 2008;Hegemann et al, 2015;Paliwal et al, 2007;Roberts et al, 2000;Zeitlinger et al, 2003). In parallel experiments using phospho-p44/ p42 antibodies, we determined that gradient tracking is the result of the distinct temporal and dose-dependent activation properties of Fus3.…”