Multi-phosphorylation reaction and clustering tune Pom1 gradient mid-cell levels according to cell size

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Abstract

Protein concentration gradients pattern developing organisms and single cells. In Schizosaccharomyces pombe rod-shaped cells, Pom1 kinase forms gradients with maxima at cell poles. Pom1 controls the timing of mitotic entry by inhibiting Cdr2, which forms stable membrane-associated nodes at mid-cell. Pom1 gradients rely on membrane association regulated by a phosphorylation-dephosphorylation cycle and lateral diffusion modulated by clustering. Using quantitative PALM imaging, we find individual Pom1 molecules bind the membrane too transiently to diffuse from pole to mid-cell. Instead we propose they exchange within longer-lived clusters forming the functional gradient unit. An allelic series blocking auto-phosphorylation shows multi-phosphorylation shapes and buffers the gradient to control mid-cell levels, which represent the critical Cdr2-regulating pool. TIRF imaging of this cortical pool demonstrates more Pom1 overlaps with Cdr2 in short than long cells, consistent with Pom1 inhibition of Cdr2 decreasing with cell growth. Thus, the gradients modulate Pom1 mid-cell levels according to cell size.

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All data generated during this study are included in the manuscript and supporting files.

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Veneta Gerganova

Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

Competing interests
The authors declare that no competing interests exist.
Charlotte Floderer

Institute of Physics, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

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The authors declare that no competing interests exist.
Anna Archetti

Institute of Physics, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-9049-3176
Laetitia Michon

Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

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The authors declare that no competing interests exist.
Lina Carlini

Institute of Physics, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

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The authors declare that no competing interests exist.
Thais Reichler

Institute of Physics, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

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The authors declare that no competing interests exist.
Suliana Manley

Laboratory of Experimental Biophysics, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

For correspondence
suliana.manley@epfl.ch

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-4755-4778
Sophie G Martin

Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

For correspondence
Sophie.Martin@unil.ch

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-5317-2557

Funding

Swiss National Science Foundation (CRSII3_160728)

Suliana Manley
Sophie G Martin

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.