(A) Mean cell length at division of the pom1 phospho-mutant allelic series in otherwise wild type and tea4Δ background with number of quantified cells indicated, *, p=10−2, **, p=10−7, ***, p=10−10, ****, p≤10−20, *****, p=10−40. (B) Correlation plot of cell length at division (values from panel A) versus Pom1 intensity at cell middle (values from Figure 4C). Error bars are standard error. (C) TIRF imaging of Pom1, shown with identical acquisition and contrasting parameters (bottom) and adjusted contrast (top). Quantification of intensities at cell middle is shown on the right (strains imaged the same day plotted together). Pom11A-GFP, Pom13A-GFP and Pom15A-GFP show increased cortical levels compared to Pom1WT-GFP (p=0.01, p=10−14, p=10−41, respectively). Note that cytoGFP expressed from the pom1 promoter strain was mixed with a Cdr2-GFP strain to identify the TIRF focal plane (green dotted line). Scale bar 2.5 μm. Images for Pom11A-GFP and Pom15A-GFP are in supplement 2. (D) Mean cluster area (left) and average cluster intensity (right) for individual Pom1 clusters from wild type versus tea4Δ background, n = 25 cells from two individual experiments. Number of clusters indicated below violin plot. The mean area is shown at the top, ****, p=10−15. (E) Localization of Cdr2-GFP and Pom1-GFP by TIRF microscopy. Left panels show a snapshot of time point 0 from a time series imaged every second for 60 s. Right panels show kymographs along the dotted line. Scale bar 2.5 μm. (F) Examples of Pom1-GFP cluster behaviors in TIRF, taken from two cells imaged every 100 ms over 8 s. Line 1: Cluster splitting (3.0 s and 4.4 s) and merging (3.2 s). Line 2: Cluster fluorescence fluctuations (down at 0.1 s, 2.0 s, 2.4 s; up at 0.2 s, 2.1 s, 2.5 s). This cluster also exhibits clear lateral movement. Line 3: Cluster fluorescence fluctuation (down at 2.4 s and 3.6 s; up at 2.6 s and 4.0 s) and disappearance (7.0 s). Line 4: Merging of two clusters (at 6.2 s). The bottom cluster exhibits clear lateral movement. Scale bars: 1 µm. (G) Minimal lifetimes of Pom1 clusters in seconds. Note that the lifetime of all but two clusters is underestimated, as they either existed at the start or end of the timelapse, or both, n = 38 clusters from 12 cells.