Extended Data Fig. 1. Colocalization of FtsN_cytoHis and FtsQ_cytoHis with FtsZ filaments depends on their interaction with FtsA.

a. Left: The surface density of FtsN_cytoHis scales linearly with density of mono-Ni-NTA (black dots) and tris-NTA-NTA lipids (red dots, mean±SD, n=3 or higher). Right: Colocalization efficiency of FtsN_cytoHis to FtsA-FtsZ filaments at increasing concentrations of mono-Ni-NTA (mean±SD, n=3 or higher).

b.(i) The FtsZ pattern is not affected at 0.2 mol% tris-Ni-NTA (or mono-Ni-NTA at 1 mol%), n=5. Scale bars are 5 μm. (ii) FtsN_cytoHis (0.5 μM) in solution does not affect the FtsZ-FtsA cofilament pattern and shows only weak binding to FtsA/FtsZ filaments, n=5. Scale bars are 10 μm. (iii) FtsQ_cytoHis shows no colocalization to FtsZ filaments (cyan) on membranes without Ni-NTA. Scale bars are 10 μm, n=3. (iv-v) FtsN_cytoHis and FtsQ_cytoHis do not colocalize with FtsZ filaments when FtsA is replaced by ZipA, n=3. Scale bars are 5 μm.

c. PBP3_cytoHis and FtsL_cytoHis attached to 0.2 mol% tris-Ni-NTA lipid membrane remained homogeneously distributed in the presence of FtsA-FtsZ filaments. Representative micrographs from n=7 (PBP3) and n=3 (FtsL) independent experiments. Scale bars are 5 μm.

d. FtsN_cytoHis does not interact with FtsZ. After addition of 0.5 μM FtsA in the same experiment FtsN_cytoHis and FtsZ are organized into cytoskeleton patterns. A similar experiment was performed with FtsQ_cytoHis, n=3. Scale bars are 5 μm.

e. Sedimentation velocity (SV) characterization of protein-detergent complexes formed by the solubilization of 16 μM full-length FtsN in 3.72 mM DDM. Sedimentation velocity c(s) distributions obtained from the analysis of the absorbance (orange line) and Raleigh interference (grey dashed line) signals of FtsN complexes using the program SEDFIT. Main peaks at 4.1 ± 0.1 and 5.2 ± 0.1 S detected by Raleigh interference (short dash line, n=3) are compatible with FtsN monomers and dimers.

f. SV analysis of AlexaFluor488FtsA in the presence of full-length FtsN at a 1:1 FtsA:FtsN molar ratio (blue) and a ~1:4 FtsA:FtsN molar ratio (red), or in the absence of FtsN (black). The main peak of FtsA at ~2 S shifts to a higher s-value upon addition of FtsN, indicating the formation of higher molecular weight species, n=2.

g. Binding curves obtained by MST of Alexa647-FtsA titrated with corresponding peptides. Binding was observed only for FtsN_cytoHis and FtsN-FL, n=3. See Extended Data 10 for fitting results.