Extended Data Fig. 7. Decrease in membrane fluidity does not affect colocalization and comigration with treadmilling FtsZ filaments.

a. The FtsZ filament pattern and colocalization with FtsN_cytoHis is not affected on low fluidity membranes (supplemented with 30% cholesterol), n=3, Scale bars are 5 μm.

b. Addition of cholesterol to a standard lipid composition slows down diffusion of FtsN_cytoHis, n=3 (bulk diffusion constant obtained from FRAP profile analysis) D_coef FtsN_cyto-His_0%Chol = 1.37 ± 0.31 D_coef FtsN_cyto-His_30%Chol = 0.90 ± 0.21. After addition of FtsA and FtsZ, FtsN_cytoHis shows the same diffusion constant on high and low fluidity membranes as diffusion is limited due to interaction with FtsZ/FtsA cofilaments. D_coef FtsN_cyto-His_0%Chol = 0.18 ± 0.12 D_coef FtsN_cyto-His_30%Chol = 0.10 ± 0.03; mean ± SD.

c Colocalization analysis reveals that a decrease in membrane fluidity does not affect colocalization efficiency between FtsN_cytoHis and FtsZ, n=5.

d. Differential kymographs, n=3 and e. treadmilling analysis (n=3) of FtsZ and FtsN co-migration on low-fluidity membranes with 30% cholesterol. Velocity histograms and fit to a Gaussian distribution (solid line) did not reveal any significant difference in the velocities of FtsZ (v = 35.6 ± 13.2 nm/s; n = 3; cyan) and FtsN (v = 37.1 ± 16.8 nm/s; n = 3; magenta) on low fluidity membranes, p-value = 0.33. The velocity values represent mean and SD from the corresponding Gaussian fits (R² = 0.97-0.99). The boxes indicate the 25–75th percentiles, whiskers the outliers, the midline indicates the median and square indicates the mean. P-value was calculated using a two-tailed Student’s t-test for parametric distributions.