Expression of GFP-tagged ScMreB5 in S. pombe cells reveals differences in filament assembly between the ATP hydrolysis mutant ScMreB5 ^E134A and ScMreB5 ^WT . (A) ScMreB5^WT and ScMreB5^E134A with N-terminal tagged GFP expressed in S. pombe cells. Representative images are shown for the DIC images of the cell boundaries (top column), a GFP channel (middle column), and overlay (bottom column). (B) 3D-SIM images of ScMreB5^WT and the ScMreB5^E134A mutant are shown (related to Video 1). Filaments of ScMreB5^WT appear as bundles spanning from one end to the other of the cell, whereas ScMreB5^E134A filaments are not tightly bundled. (C) Plot showing the anisotropy, an indicator of parallelness of filament arrays, of ScMreB5^WT and the ScMreB5^E134A mutant. Plots are shown as superplots from three biological replicates (N = 3) with number of cells measured in each replicate ≥23 and ≤112. (D and E) Plots showing CV (D), a metric indicating the extent of filament bundling, and density (E), which is the amount of ScMreB polymers per unit area of cells, of ScMreB5^WT and the ScMreB5^E134A mutant. Plots are shown as superplots from three biological replicates (N = 3) with number of cells measured in each replicate being ≥23 and ≤93. (F) Plot comparing the percentage of cells showing either diffuse fluorescence or filaments for ScMreB5^WT and ScMreB5^E134A. Cells were grown in EMM without thiamine for 36–40 h. Cells expressing ScMreB5^E134A show more diffused fluorescence and very few filaments in comparison to ScMreB5^WT. The mean values (N = 3) are plotted, and the error bar denotes mean with SEM. Fisher’s exact test, two-tailed. (G) Plot showing the mean fluorescence intensity of ScMreB5^WT and the ScMreB5^E134A mutant cells having diffuse fluorescence. The average diffuse fluorescence intensities in ScMreB5^E134A mutant cells are higher than in ScMreB5^WT, suggesting a higher critical concentration of polymerization for the ATPase mutant ScMreB5^E134A. Mean fluorescence intensities were calculated from sum intensity projections of cells with diffuse fluorescence. Plots are shown as superplots from three biological replicates (N = 3) with number of cells measured in each replicate being ≥23 and ≤112. For each replicate in C–E and G, ScMreB5^WT and the ScMreB5^E134A were grown at the same time to account for day-to-day variations in the growth of cultures at the single-cell level. Statistical significance was assessed by paired two-tailed Student’s t test. P values were calculated using Microsoft Excel formula TTEST. The error bars shown are inferential and represent 95% CI. The mean values of each replicate in the superplot for ScMreB5^WT and ScMreB5^E134A are connected by dotted lines.