MamK filament growth behavior analysis by FRAP and photoconversion. a Kymographs displaying fluorescence signal intensity (x-axis) of bleached MamK filaments over the time (y-axis). mCherry-MamK fusion expressed from a plasmid (P
promoter) in MSR: (ai) wildtype (WT), (aii) ∆mamK, (aiii) ∆mamJK, and (aiv) MSR-1B. The corresponding duplicated kymograph indicates bleaching time/area (red box) and filament fluorescent signal progression (white dashed line). The bleach-marked filaments were followed for 5 min (imaging every 30 s). bi Photoconversion of Dendra2-MamK expressed in WT cells from a plasmid (P
promoter). Green channel: MamK filament prior to photoconversion. Red channel: photoconverted protein after a 405 nm laser pulse. White dashed circle: photoconverted area. White dashed lines act a reference point. Red dashed lines: filament growth progression. Arrow indicates appearance of MamK signal at the cell pole. A total of 91 % of cells (n = 54) showed polar appearance of the photoconverted protein. bii Cartoon illustrating intracellular Dendra2-MamK dynamics. After the laser pulse (left pole), the filament and Dendra2-MamK free subunits are photoconverted (green-to-red). Freely diffusible Dendra2-MamK subunits migrate and incorporate at the right cell pole, where MamK filaments originate. biii Kymograph of a cell (Additional file 18: Figure S11E) displaying intracellular localization of the red photoconverted signal (x-axis) over time (y-axis). The corresponding duplicated kymograph below indicates the photoconverted time/area (red box) and MamK filament fluorescent signal appearance/progression (red dashed lines). biv Quantification of photoconverted Dendra2-MamK signal in WT cells (n = 21). c Photoconversion of Dendra2-MamKD161A as in “bi”. Lack of dynamics was observed in 100 % of cells (n = 16) (see Additional file 18: Figure S11D for quantitative data). d MamK filament treadmilling speed per strain quantified from mCherry-MamK photobleaching data. An unpaired Student’s t-test was performed. * Significant: P = 0.01 to 0.05. ** Very significant P = 0.001 to 0.01. *** Extremely significant P < 0.001. ns: not significant.