Supplementary MaterialsFIG?S1. Brequinar inhibition of 1 1.5. Download FIG?S1, TIF file, 1.1 MB. Copyright ? 2019 Billaudeau et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. TABLE?S1. Strains used in this study. Download Table?S1, TIF file, 0.2 MB. Copyright ? 2019 Billaudeau et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. MOVIE?S1. TIRF acquisitions of strains expressing under an inducible promoter (RCL238), grown to exponential (Expo) or stationary (Stat) growth phase in the presence of 0.5% xylose. Images of cells in exponential and stationary phase were taken every second for 1 min and every other second for 2 minutes, respectively (thus, the stat record appears accelerated 2 compared to expo). Download Movie S1, AVI file, 0.1 MB. Copyright ? 2019 Billaudeau et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S2. (A) Length quantification of long filaments using a kymograph. In panel 1, by TIRF or SIM-TIRF, Brequinar inhibition directionally moving MreB particles appear as discrete, light diffraction-limited dots (a) or filamentous structures (b). In panel 2, profile lines (1-pixel width) were drawn along separated filament trajectories (red lines) in order to generate kymographs. In panel 3, on kymographs, slices are piled up vertically: the distance (strain (RCL238) grown to exponential phase, quantified using the kymograph (green; = 31,376) method. (C) Determination of the length of simulated filaments shows the requirement of a kymograph approach to measure long structures. Objects ranging from 100 to 1 1,300 nm long were simulated and measured using three methods: kymograph (black), 1D Gaussian fit of intensity profile (red), or 1D Gaussian fit of intensity profile with a simulated intensity decrease due to TIRF illumination (magenta). For this, a correction factor to the intensity exp(?of filament relative to the coverslip, where is the cell radius and is the lateral distance separating the filament position from the contact point of the cell with the coverslip (inset). Error bars correspond to standard deviations (strains during exponential growth. The lengths shown are the median values detected along their track and were determined by the G-fit method. = Il1a 13,070 (NC103), 22,053 (RCL238), 13,666 (JS17), and 4,625 (2521). (E) Distribution of length of GFP-MreB (RCL238, JS17, and NC103) and Mbl-GFP (2521) fusions in strains during stationary phase. Lengths are estimated on directionally moving subpopulation of filaments by the kymograph method (see Materials and Methods). = 37 (RCL238), 35 (JS17), 38 (NC103), and 85 (2521). (F) Example of micron-long GFP-MreB artifacts forming during stationary phase. Cells of the merodiploid JS17 strain were grown in rich LB medium and observed 2 h after entry into stationary phase. A slice of a 3D SIM-TIRF acquisition corresponding to Movie S3 is shown here. Download FIG?S2, TIF file, 0.6 MB. Copyright ? 2019 Brequinar inhibition Billaudeau et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. MOVIE?S2. TIRF and SIM-TIRF comparison of acquisitions on strains expressing under an inducible promoter (RCL238), grown to exponential phase in the presence of 0.5% xylose. Images were taken every second for 1 min and processed with false color to enhance the visualization. Bars = 0.5 m. Download Movie S2, AVI file, 0.3 MB. Copyright ? 2019 Billaudeau et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. MOVIE?S3. Example of.