Editors' ChoiceCell Size

Shaping the Gradient

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Science Signaling  28 Jun 2011:
Vol. 4, Issue 179, pp. ec178
DOI: 10.1126/scisignal.4179ec178

Cortical concentration gradients of the kinase Pom1 link cell size to mitosis in rod-shaped fission yeast: Bipolar Pom1 gradients originating from the cell tips extend far enough in short—but not long—cells to inhibit an inducer of mitosis located in the cortex at the cell equator. Noting that Pom1 localization depends on its kinase activity and on the microtubule-associated cell-tip protein Tea4, Hachet et al. used fluorescently labeled proteins to explore the mechanisms underlying the establishment of Pom1 gradients. Loss of Tea4 essentially abolished Pom1 localization to the cell cortex. The distributions of Tea4 and Pom1 were distinct, with cortical Pom1 spreading further from the tip, and fluorescence recovery after photobleaching analysis indicated that Pom1 moved laterally along the plasma membrane. Ectopic localization of Tea4 to foci in the lateral cortex led to Pom1 recruitment to these foci, from which it also spread along the membrane. Analyses of Pom1 truncation mutants indicated that its cortical localization depended on a region with abundant charged lysine and arginine residues; moreover, the Pom1 N terminus (Pom11-699, which contained this region) bound phosphatidylserine and phosphatidylinositol phosphates (negatively charged components of the plasma membrane). A catalytically inactive form of Pom1 (Pom1KD) spread throughout the cortex, as did a mutant form of Pom1 in which serines or threonines were substituted with alanines at six candidate autophosphorylation sites external to the kinase domain (Pom16A). In vitro kinase assays and assessment of its SDS-PAGE migration pattern indicated that Pom1 was indeed autophosphorylated, an observation substantiated by mass spectrometric analysis. Unphosphorylated Pom1 resided more stably at the membrane than phosphorylated Pom1; moreover, dephosphorylation enhanced Pom1 binding to membrane lipids. Tea4 bound Pom1, an interaction implicated in Pom1’s localization to the cortex; unexpectedly, however, Pom1KD and Pom16A localized to the cortex of cells lacking Tea4 or containing a Tea4 mutant defective for Pom1 binding. Tea4 recruits the phosphatase Dis2 to cell tips, and bimolecular fluorescence complementation indicated that Pom1, Tea4, and Dis2 were in close proximity, with the proximity of Pom1 and Dis2 depending on Tea4. Moreover, localization of Pom1 to the cell tip was decreased by disruption of Dis2 binding to Tea4 and abolished by disruption of Tea4 binding to both Dis2 and Pom1. The authors propose that dephosphorylation of Pom1 by Dis2 exposes a positively charged region, initiating its binding to the cell membrane; autophosphorylation decreases its association with the membrane, allowing it to diffuse into the cytoplasm and thereby limiting lateral movement in the cortex. Thus, Pom1 autophosphorylation, in combination with its lateral diffusion along the membrane and its dephosphorylation by Dis2, shapes the Pom1 cortical concentration gradient.

O. Hachet, M. Berthelot-Grosjean, K. Kokkoris, V. Vincenzetti, J. Moosbrugger, S. G. Martin, A phosphorylation cycle shapes gradients of the DYRK family kinase Pom1 at the plasma membrane. Cell 145, 1116–1128 (2011). [PubMed]

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