Extended Data Fig. 5: FRAP analysis.
a. Micrographs of FRAP experiments (top) and corresponding intensity profiles across photobleached regions of Cy5-FtsZ (left, cyan, n = 5), TMR-FtsA (middle, green, n = 3) and CF488-FtsNcytoHis (right, magenta, n = 6) at different time points. The first micrograph shows the area before bleaching, micrographs at 0 s correspond to the pattern at the first frame after bleaching. Scale bars are 10 µm. Fitting a Gaussian error function to the profiles to obtain 𝜎2(t) points reveals changes in the profile shape over time (inset plots). For FtsZ, 𝜎2 remains constant, showing that fast recovery of the photobleached region is dominated by a homogenous exchange of FtsZ monomers instead of lateral diffusion. For FtsA and FtsNcytoHis, 𝜎2 increases with time, consistent with a strong contribution of lateral diffusion for recovery. Shaded areas represent the standard deviation. This can also be seen by the delayed recovery of fluorescence in the center of the bleached area compared to its edges. See Supplementary Video 7. b. Recovery of FtsNcytoHis and rhodamine-DOPE are comparable, within the error bar, in the absence of FtsA and FtsZ. The recovery of FtsNcytoHis (magenta) within the rectangular profile is slowed down significantly after FtsA and FtsZ have been added (FtsN alone: T0.5 = 14.9 ± 2.6 s, mean ± s.d., FtsN + FtsA/FtsZ: T0.5 = 75.4 ± 25.6 s (n = 4)), whereas rhodamine recovery (grey) decreased slightly, presumably due to crowding effects by membrane-attached FtsZ-FtsA co-filaments. (rhodamine-PE: T0.5 = 12.8 ± 3.4 s, rhodamine-PE + FtsA/FtsZ: T0.5 = 17.7 ± 0.2 s (n = 3)). Scale bars are 10 µm. Line represents the mean, error bars the standard deviation.
