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. 2019 Jan 21;10:268. doi: 10.1038/s41467-018-08218-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Rga4-dependent rescaling of cellular geometry is essential for medial division plane positioning. a–c Single z-plane bright-field micrographs of S. japonicus cells of indicated genotypes incubated with 20 μM 3-BrB-PP1 (right) for 2 h. Note that Rga4 deficiency prevents hyperpolarization of growth upon Wee1 inhibition. d Quantifications of cell diameters in the “thinner” (top) and “thicker” daughter (bottom) cell at cytokinesis, when 3-BrB-PP1 treatment is combined with genetic disruption of Cdc42 module regulators shown in (a–c). e wee1-as8 rga4Δ cells incubated with 20 μM 3-BrB-PP1 (right) for 4 h show severe division site positioning defects. Shown are bright-field micrographs. f Quantifications of cellular aspect ratio at division of cells shown in (e). g Single z-plane spinning-disk confocal micrographs of Pom1-GFP-expressing rga4Δ and wee1-as8 rga4Δ cells treated with 20 μM 3-BrB-PP1 for 2 h. h A plot summarizing the accuracy of division plane positioning in cells shown in (e). Deviation from the geometric center of the cell is indicated on y-axis. Black bars represent sample median. i Time-lapse montage of maximum intensity z-projected spinning-disk confocal micrographs of 3-BrB-PP1-treated wee1-as8 rga4Δ S. japonicus cells expressing Rlc1-GFP and the nucleoplasmic protein Nhp6-mCherry. Cell boundaries are outlined by white dashed lines. Wee1-inhibited S. japonicus cells lacking Rga4 fail to anchor the actomyosin ring medially following the semi-open mitosis, as indicated by the dispersal and nuclear re-import of Nhp6. a–c, e, g, i Scale bars represent 5 μm. d, f, h n indicated in figures; p values derived using Kolmogorov–Smirnov test