Time-lapse imaging of GFP-tagged ScMreB5 in S. pombe cells for estimating the differences in filament assembly dynamics of the ATP hydrolysis mutant ScMreB5 ^E134A . (A) Time-lapse microscopy showing polymerization for ScMreB5^WT and ScMreB5^E134A (related to Video 2). Cells were grown for 28–30 h in the absence of thiamine, placed on an agarose pad without thiamine as mentioned in Materials and methods, and imaged at every 3-min time interval. The time at which the cells were placed on agarose pads and first imaged was taken as t = 0. (B) Plot showing the difference in time lag in the polymerization of ScMreB5^E134A (number of cells = 7) compared with ScMreB5^WT (number of cells = 9). In cells where polymerization was observed, it was seen to initiate between 6 to 57 min, with a mean time of ∼26 min for ScMreB^WT as mentioned in the text. For ScMreB^E134A mutant, the time was significantly delayed, and polymerization was observed between 42 to 93 min, with a mean of 69 min. Error bars represent 95% CI; statistical significance was assessed using an unpaired two-tailed Student’s t test, and the P value (0.00044) was calculated using the formula TTEST in Microsoft Excel. (C) A time-course experiment showing the lag in polymerization of ScMreB5^E134A compared with ScMreB5^WT. The percentage of cells (number of cells counted for each biological replicate was ≥1,239 and ≤1,792) having filaments were calculated for ScMreB5^WT and ScMreB5^E134A and plotted as a function of time. The mean from three biological replicates (N = 3) is plotted, and the error bars are inferential and represent 95% CI. (D) A representative immunoblot (one of three repeats) showing similar levels of protein expression for ScMreB5^WT and ScMreB5^E134A. ScMreB5^WT and ScMreB5^E134A were detected using anti-GFP antibodies conjugated to HRP, and β-tubulin was used a loading control. Band intensities were measured using Fiji, and the numbers below the GFP blot indicate values normalized with respect to the tubulin band. (E) Time-lapse microscopy showing bundling for ScMreB5^WT and defect in bundling for ScMreB5^E134A filaments (related to Video 3). White arrows indicate bundling events in ScMreB5 and, for ScMreB5^E134A, point to the site of septation and highlight the bundling events that happen at the time of cell division (8 cells out of 41). (F) Time-lapse microscopy of cells expressing ScMreB5^WT, which are undergoing division (related to Video 4). Filament disassembly can be observed after cell division. (G) Time-lapse microscopy of cells expressing ScMreB5^WT shows fragmentation and reannealing or bundling of filaments (related to Video 5). Scale bar denotes 5 µm. For A, B, F, and G, cells were grown in EMM without thiamine for 24–32 h before imaging. Cells were placed on an EMM agarose pad lacking thiamine as described in Materials and methods and imaged at every 3-min time interval. For E–G, the number of cells in which such events were observed and the total number of cells are indicated in parentheses. Source data are available for this figure: SourceData F5.