Design principles of Cdr2 node patterns in fission yeast cells

Abstract

Pattern forming networks have diverse roles in cell biology. Rod-shaped fission yeast cells use pattern formation to control the localization of mitotic signaling proteins and the cytokinetic ring. During interphase, the kinase Cdr2 forms membrane-bound multiprotein complexes termed nodes, which are positioned in the cell middle due in part to the node inhibitor Pom1 enriched at cell tips. Node positioning is important for timely cell cycle progression and positioning of the cytokinetic ring. Here, we combined experimental and modeling approaches to investigate pattern formation by the Pom1-Cdr2 system. We found that Cdr2 nodes accumulate near the nucleus, and Cdr2 undergoes nucleocytoplasmic shuttling when cortical anchoring is reduced. We generated particle-based simulations based on tip inhibition, nuclear positioning, and cortical anchoring. We tested model predictions by investigating Pom1-Cdr2 localization patterns after perturbing each positioning mechanism, including in both anucleate and multinucleated cells. Experiments show that tip inhibition and cortical anchoring alone are sufficient for the assembly and positioning of nodes in the absence of the nucleus, but that the nucleus and Pom1 facilitate the formation of unexpected node patterns in multinucleated cells. These findings have implications for spatial control of cytokinesis by nodes and for spatial patterning in other biological systems.

Fig 1:. Regulated nuclear shuttling of Cdr2.

(A) Single middle focal plane images of Cdr2 and the nuclear marker Cut11 in the indicated strains after leptomycin B (LMB) treatment. Scale bar, 5 μm. (B) Quantification of Cdr2 nuclear signal after LMB. $\mathrm{n}=25$ cells. ns >0.05, *** ≤0.001, **** ≤0.0001 by one-way ANOVA with Tukey’s multiple comparison test. (C) Single middle focal plane images of Cdr2 node localization in skinny (rga2Δ) and wide (rga4Δ) mutants. White arrows indicate off-center nuclei. Asterisks mark mitotic cells with diffuse cytoplasmic Cdr2. Scale bar, 5 μm. (D). (D) Quantification of Cdr2 node intensity ratio for the indicated strains. $\mathrm{n}>25$ cells. * ≤0.05, *** ≤0.001, **** ≤0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. (E) Schematic of particle simulation model. Color of arrows indicates activation (red), inactivation (purple), or translocation (black). (F) Snapshots Cdr2 distribution after 50 min equilibration, for wild type, arf6Δ, and anucleate cell cases. Area and intensity of Cdr2 nodes (green) proportional to number of molecules per node. Pom1: magenta. Cell length: 11 μm.

Fig 2:. Cdr2 nodes follow displaced nuclei.

(A) Start of nuclear displacement simulation after equilibrating Cdr2 for 50 min.. (B) Simulated response to nuclear movement with node area and color according to number of Cdr2 molecules. Asterisks mark the cell middle. (C) Single middle focal plane images of Cdr2 and Mid1 before and 15 minutes after nuclear displacement. Boxes show enlarged images of node region highlighted with a yellow dashed box. Strains are cdc25degron-DAmP (cdc25-dD) mutation to increase cell length. Scale bar, 5 μm. (D) Line scans of the boxed regions in panel C of Cdr2 and Mid1 at the highlighted node region. Cdr2 and Mid1 colocalize in nodes before and after movement of the nucleus. (E) Quantification of Cdr2 fluorescence at the cell center or near the tip in cells before and after nuclear movement. $\mathrm{N}=10$ cells *** ≤0.001, **** ≤0.0001 by one-way ANOVA with Dunnett’s multiple comparison test.

Fig 3:. Formation of Arf6-dependent nodes in anucleate cells.

(A) Maximum projection images of cells at the indicated timepoints after centrifugation. Images show anucleate and binucleate cells, with anucleate cell noted by the white asterisk. Cells are cdc25-dD to increase cell length. Scale bar 5 μm. (B) Simulated distribution of Cdr2 nodes for a mononucleate and anucleate cell after 50 min equilibration. (C) Fluorescence intensity of individual nodes 60 minutes after spin. $\mathrm{n}>10$ cells each. **** ≤ 0.0001. (D) The number of Cdr2 nodes per cell 60 minutes after spin. $\mathrm{n}>10$ cells. * ≤0.05, ** ≤0.01. (E) Arf6 is required for Cdr2 nodes in anucleate cells. Maximum projection image of cells 75 minutes after centrifugation. Anucleate cell labeled A, bincleate cell labeled B, dividing cell with diffuse cytoplasmic Cdr2 labeled with asterisk. Scale bar 5 μm. (F) The number of nodes per arf6Δ cell 60 minutes after spin. $\mathrm{n}>10$ cells. **** ≤0.0001. (G) Quantification of cytoplasmic Cdr2 levels in arf6Δ cells 60 minutes after spin. $\mathrm{n}>10$ cells. **** ≤0.0001. All statistical tests in this figure are Welch’s unpaired t-test.

Fig 4:. Spatial patterning of Cdr2 nodes in multinucleated cells.

(A) Localization of Cdr2-mEGFP nodes in representative multinucleated cells. Nuclei and cell boundaries are marked by ER protein Sur4-mCherry. Images are maximum intensity projections of a Z-series. (B) Single middle focal plane images of multinucleated cells. Hoechst stain marks nuclei. Line scans of Cdr2 (green) and Pom1 (magenta) are shown below each image. Scale bars, 5 μm. (C) Working model for positioning of Cdr2 nodes by overlapping positive and negative signals.