Extended Data Fig. 6: Single-molecule analysis of FtsN_cyto-TM in supported bilayers supports a diffusion-and-capture mechanism.

a. Schematic illustration of the reconstitution approach: i. FtsN_cyto-TM was incorporated into SUVs by detergent extraction; ii. Proteoliposomes were adsorbed on a surface modified with a dense film of PEG2000-palmitic acid. Vesicle rupturing and formation of polymer-SLB were induced by addition of PEG 8 kDa. iii. FtsZ and FtsA are then allowed to self-organize on the membrane surface. b. Micrographs showing colocalization of FtsN_cyto-TM-Cy5 with Alexa Fluor 488-FtsZ. An FtsN_cyto-TM pattern similar to FtsZ was observed after addition of FtsZ and FtsA confirming recruitment of the transmembrane peptide to FtsZ-FtsA filaments, n = 2. To enhance visualization of the filament bundles, images were processed by subtracting an averaged background over a circular area of 20 px, scale bars are 10 µm. c. Micrographs and detected trajectories of single FtsN_cyto-TM peptides alone, in the presence of 1.5 µM FtsZ/ 0.5 µM FtsA (n = 2) and with of 3 µM FtsZ/ 0.5 µM FtsA (n = 1). Scale bars are 10 µm for micrographs and 5 µm for corresponding trajectory maps. d. Left: Quantification of the diffusion coefficients of single molecules reveals presence of two populations for FtsN_cyto-TM (n = 2 videos, N = 2 experiments), indicative for non-specifically stuck particles. Addition of FtsA and FtsZ slows down mobility of fast diffusing population (left). Right: Box plot of obtained diffusion constants. Although FtsN_cyto-TM (n = 2, N = 2) diffuses about 2-fold slower than FtsN_cytoHis (n = 2, N = 4), their diffusivities were comparable after addition of FtsZ and FtsA (left). The diffusion coefficients are 0.70 ± 0.14 µm²/s for FtsN_cyto-His and 0.34 ± 0.21 µm²/s for FtsN_cyto-TM without FtsA/FtsZ; 0.15 ± 0.04 µm²/s for FtsN_cyto-His and 0.21 ± 0.07 µm²/s for FtsN_cyto-TM in the presence of FtsA and FtsZ; (values are mean ± s.d.). The boxes indicate the 25–75th percentiles, whiskers the outliers, the midline indicates the median and square indicates the mean. e. Directional autocorrelation analysis confirms random non-correlated behavior of single FtsN_cyto-TM molecules at 1.5 µM (left) and 3.0 µM (right) FtsZ, from all trajectories in one representative experiment. Line represents mean, error bars the standard deviation. f. Confinement time analysis of FtsN_cyto-TM (0.66 ± 0.1 s, mean ± s.d., n = 1, N = 3) shows similar confinement time as FtsN_cyto-His.

Source data