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Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling

Nature Cell Biology volume 9, pages 1319–1326 (2007)Cite this article

Abstract

Signal transduction through mitogen-activated protein kinase (MAPK) cascades is thought to occur through the assembly of macromolecular complexes. We quantified the abundance of complexes in the cytoplasm among the MAPKs Ste11, Ste7, Fus3 and the scaffold protein Ste5 in yeast pheromone signalling using fluorescence cross-correlation spectroscopy (FCCS). Significant complex concentrations were observed that remained unchanged on pheromone stimulation, demonstrating that global changes in complex abundances do not contribute to the transmission of signal through the cytoplasm. On the other hand, investigation of the distribution of active Fus3 (Fus3PP) across the cytoplasm using fluorescence lifetime imaging microscopy (FLIM) revealed a gradient of Fus3PP activity emanating from the tip of the mating projection. Spatial partitioning of Fus3 activating kinases to this site and deactivating phosphatases in the cytoplasm maintain this Fus3PP-activity distribution. Propagation of signalling from the shmoo is, therefore, spatially constrained by a gradient-generating reaction-diffusion mechanism.

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Figure 1: Localization of endogenously expressed Ste5, Ste7, Ste11 and Fus3 in vegetative and pheromone-stimulated cells.
Figure 2: Quantification of complexes between Ste5, Ste7, Ste11 and Fus3 in the cytoplasm through measurement of all six pair-wise interactions.
Figure 3: Recruitment of active Fus3PP to the shmoo.
Figure 4: Cytoplasmic phosphatase levels influence the binding of Fus3 at the shmoo.
Figure 5: A gradient of active Fus3PP emanates from the shmoo across the cytoplasm.

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Acknowledgements

We would like to thank R. Pepperkok, T. Zimmermann, J. Rietdorf, S. Terjung and A. Seitz of the advanced light microscopy facility (ALMF) of EMBL and L. Kuschel (Leica Microsystems, Germany) for their continuous support, and our reviewers for constructive comments. G. F. Fink is acknowledged for plasmid pBI479 (PFUS1–lacZ) and W. A. Lim for plasmids containing STE7 and STE5 mutants.

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Author notes

  1. Reinhard Mayr

    Present address: Current address: Upper Austrian Research GmbH, Center for Biomedical Nanotechnology (CBN), Scharitzerstraße 6-8, 4020 Linz, Austria.,

  2. Celine I. Maeder and Mark A. Hink: These authors contributed equally to this work.

Authors and Affiliations

  1. Cell Biology and Biophysics Unit, EMBL-Heidelberg, Meyerhofstrasse 1, D-69117, Heidelberg, Germany

    Celine I. Maeder, Reinhard Mayr & Michael Knop

  2. Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227, Dortmund, Germany

    Mark A. Hink, Ali Kinkhabwala & Philippe I. H. Bastiaens

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  1. Celine I. Maeder
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Correspondence to Philippe I. H. Bastiaens or Michael Knop.

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Supplementary figures S1 to S6, Supplementary materials and methods (PDF 1842 kb)

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Maeder, C., Hink, M., Kinkhabwala, A. et al. Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling. Nat Cell Biol 9, 1319–1326 (2007). https://doi.org/10.1038/ncb1652

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