Wee1 accumulation at nodes is size dependent and buffered by Pom1. (A) Representative images of Wee1 node localization in cells of increasing size imaged by TIRF microscopy. Bar, 5 µm. (B) Quantification of the number of Wee1 puncta as a function of cell size. Values were obtained from TIRF images with 1-s exposure. Data fit a linear regression model. (C) Wee1 burst duration scales with cell size in WT but not cdr2(E177A) or pom1Δ cells. The duration of individual Wee1-mNeonGreen bursts was measured using time-lapse TIRF microscopy for cells of the indicated genotype. Dots correspond with mean burst duration plotted as a function of cell size. Lines are linear regressions fit to these data. Dotted lines indicate the 95% confidence interval of the linear regression accounting for SD and sample size of the mean for each data point. The resulting slope is significantly nonzero (m ≠ 0) for WT (P = 0.002; R² = 0.02) but not for pom1Δ (P = 0.01; R² = 0.01) or cdr2(E177A) (P = 0.9; R² = 3.3e⁻⁷). (D) Pom1 suppresses Wee1 bursts in small cells. The number of Wee1 puncta was quantified as in B for WT and pom1Δ cells, and the data were binned according to cell size. Boxes indicate means and SD, and whiskers indicate minimum and maximum values. The number of Wee1 bursts is significantly different between WT and pom1Δ in the three smallest bins (7 µm: *, P = 0.01; 8 µm: ****, P < 0.0001; 9 µm: ***, P = 0.0003; 10–13 µm: P > 0.5). The 6-µm bin contains only pom1Δ cells (n_{bin(WT/pom1Δ)}) = 6 µm (0/5), 7 µm (4/10), 8 µm (28/23), 9 µm (34/24), 10 µm (32/25), 11 µm (17/21), 12 µm (21/15), and 13 µm (21/21). (E) Relative accumulation of Wee1 puncta and Cdr2 nodes in WT and pom1Δ cells as a function of cell size. Linear regressions and 95% confidence intervals are shown. The slope of the linear regression is significantly nonzero for WT (P < 0.0001; R² = 0.98) but not for pom1Δ (P = 0.7; R² = 0.26).