title: 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, cell-cycle 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 KDof 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 long-term developmental fates in a unicellular differentiation system.
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; Yale Systems Biology Institute, Yale University, West Haven, CT 06516; Department of Biomedical Engineering, Yale University, New Haven, CT 06511; andre.levchenko@yale.edu patrick.conlon@yale.edu.
authors.forename: Patrick
authors.initials: P
authors.lastname: Conlon
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; Yale Systems Biology Institute, Yale University, West Haven, CT 06516; Department of Biomedical Engineering, Yale University, New Haven, CT 06511.
authors.forename: Rita
authors.initials: R
authors.lastname: Gelin-Licht
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
authors.forename: Ambhighainath
authors.initials: A
authors.lastname: Ganesan
authors.affiliation: Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; Yale Systems Biology Institute, Yale University, West Haven, CT 06516; Department of Biomedical Engineering, Yale University, New Haven, CT 06511; andre.levchenko@yale.edu patrick.conlon@yale.edu.
authors.forename: Andre
authors.initials: A
authors.lastname: Levchenko
mesh_terms: D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D016764:Cell Polarity / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010675:Pheromones / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D059008:Time-Lapse Imaging
chemical_list: D010675:Pheromones; D029701:Saccharomyces cerevisiae Proteins; D020928:Mitogen-Activated Protein Kinases
keywords: Fus3; MAPK dynamics; cell signaling; mating pathway; yeast