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Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins

Nature Microbiology volume 5, pages 407–417 (2020)Cite this article

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Abstract

Most bacteria accomplish cell division with the help of a dynamic protein complex called the divisome, which spans the cell envelope in the plane of division. Assembly and activation of this machinery are coordinated by the tubulin-related GTPase FtsZ, which was found to form treadmilling filaments on supported bilayers in vitro1, as well as in live cells, in which filaments circle around the cell division site2,3. Treadmilling of FtsZ is thought to actively move proteins around the division septum, thereby distributing peptidoglycan synthesis and coordinating the inward growth of the septum to form the new poles of the daughter cells4. However, the molecular mechanisms underlying this function are largely unknown. Here, to study how FtsZ polymerization dynamics are coupled to downstream proteins, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ–FtsA filaments, but despite their directed collective behaviour, individual peptides showed random motion and transient confinement. Our work suggests that divisome proteins follow treadmilling FtsZ filaments by a diffusion-and-capture mechanism, which can give rise to a moving zone of signalling activity at the division site.

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Fig. 1: FtsZ–FtsA filaments colocalize with cytoplasmic tails of FtsN and FtsQ.
Fig. 2: Cytoplasmic tails of FtsN and FtsQ co-migrate directionally with treadmilling FtsZ–FtsA cofilaments.
Fig. 3: FtsNcytoHis follows treadmilling FtsZ–FtsA cofilaments via a diffusion-and-capture mechanism.
Fig. 4: Schematic of coupling between treadmilling FtsZ filaments and cell division proteins.

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Data availability

Source data for Figs. 1–3 and Extended Data Figs. 1–8 are provided with the paper. Raw data for Supplementary Videos 1–11 are available from Figshare at https://doi.org/10.6084/m9.figshare.10282046.v1. All other data supporting the findings of this study are available from the corresponding author upon request.

Code availability

The Fiji macro used for differential image processing, as well as the MATLAB codes for treadmilling velocity analysis, lifetime analysis and FRAP profile analysis, are available upon request from the corresponding author. Code source files for our treadmilling analysis are available online at https://github.com/paulocaldas/Treadmilling-Speed-Analysis.

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Acknowledgements

We acknowledge members of the Loose laboratory at IST Austria for helpful discussions—in particular, P. Caldas for help with the treadmilling analysis, M. Jimenez, A. Raso and N. Ropero for providing Alexa Fluor 488- and Alexa Fluor 647-labelled FtsA for the MST and analytical ultracentrifugation experiments. We thank C. You for providing the DODA-tris-NTA phospholipids, as well as J. Piehler and C. Richter (Department of Biology, University of Osnabruck, Germany) for the SLIMfast single-molecule tracking software and help with the confinement analysis. We thank J. Errington and H. Murray (both at Newcastle University, UK) for critical reading of the manuscript, and J. Brugués (MPI-CBG and MPI-PKS, Dresden, Germany) for help with the MATLAB programming and reading of the manuscript. This work was supported by the European Research Council through grant ERC-2015-StG-679239 to M.L. and grants HFSP LT 000824/2016-L4 and EMBO ALTF 1163-2015 to N.B., a grant from the Ministry of Economy and Competitiveness of the Spanish Government (BFU2016-75471-C2-1-P) to C.A. and G.R., and a Wellcome Trust Senior Investigator award (101824/Z/13/Z) and a grant from the BBSRC (BB/R017409/1) to W.V.

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Authors and Affiliations

  1. Institute for Science and Technology Austria, Klosterneuburg, Austria

    Natalia Baranova, Philipp Radler, Mar López-Pelegrín & Martin Loose

  2. Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK

    Víctor M. Hernández-Rocamora & Waldemar Vollmer

  3. Centro de Investigaciones Biológicas, Madrid, Spain

    Carlos Alfonso & Germán Rivas

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Contributions

N.B. and M.L. conceptualized the experiments. N.B. and P.R. performed and analysed most of the biochemical experiments. P.R. and M.L. performed the treadmilling analysis. N.B. performed the single-molecule analysis. M.L.-P., N.B., P.R. and V.M.H.-R. purified the proteins. M.L. performed the structural modelling. V.M.H.-R. performed and analysed the MST experiments. C.A. performed and analysed the sedimentation velocity and analytical ultracentrifugation experiments. N.B., P.R. and M.L. wrote the original draft. N.B., P.R., V.M.H.-R., W.V., M.L.-P. and M.L. reviewed and edited the manuscript. N.B., G.R., W.V. and M.L. supervised the study. Funding was acquired by W.V. and M.L.

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Correspondence to Martin Loose.

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Extended data

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

a. Left: The surface density of FtsNcytoHis scales linearly with density of mono-Ni-NTA (black dots) and tris-Ni-NTA lipids (red dots, mean ± s.d., n = 3 or higher). Right: Colocalization efficiency of FtsNcytoHis to FtsA-FtsZ filaments at increasing concentrations of mono-Ni-NTA (mean ± s.d., 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) FtsNcytoHis (0.5 µM) in solution does not affect the FtsZ-FtsA cofilament pattern and shows only weak binding to FtsZ-FtsA filaments, n = 5. Scale bars are 10 µm. (iii) FtsQcytoHis shows no colocalization to FtsZ filaments (cyan) on membranes without Ni-NTA. Scale bars are 10 µm, n = 3. (iv-v) FtsNcytoHis and FtsQcytoHis do not colocalize with FtsZ filaments when FtsA is replaced by ZipA, n = 3. Scale bars are 5 µm. c. PBP3cytoHis and FtsLcytoHis 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. FtsNcytoHis does not interact with FtsZ. After addition of 0.5 µM FtsA in the same experiment FtsNcytoHis and FtsZ are organized into cytoskeleton patterns. A similar experiment was performed with FtsQcytoHis, 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 Alexa Fluor 488-FtsA 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 Alexa Fluor 647-FtsA titrated with corresponding peptides. Binding was observed only for FtsNcytoHis and full-length FtsN (FtsN-FL), n = 3. See Extended Data 10 for fitting results.

Source data

Extended Data Fig. 2 Binding between FtsN and FtsA is dominated by electrostatic interactions.

a. Binding curves obtained from the MST thermograms of Alexa Fluor 647 FtsA titrated with full length FtsN at 100 (black symbols), 150 (green symbols) or 300 mM KCl (red symbols). The binding curves at the lower ionic strengths are biphasic with a first transition from 100 to 1000 nM FtsN and a second transition that could not be saturated. The interaction was significantly weaker in the presence of 300 mM KCl (red symbols). The Hill equation, with n fixed to 1 was used to calculate the midpoint of the first transition for the curves at 100 mM (black line) or 150 mM KCl (green line), resulting in apparent KD values of 250 ± 51 nM at 100 mM KCl or 734 ± 23 nM at 150 mM KCl, n = 3. b. The mutant peptides D5N (n = 5), RAAK (n = 5), D5N-RAAK (n = 3) efficiently colocalized with the FtsZ filaments similar to the wildtype peptide, while FtsNcytoDDEEHis (n = 5) mutant and the scrambled FtsN peptide (n = 6) show only weak colocalization Scale bars are 5 µm. c. Sequences of tested peptides. Dashed boxes correspond to the conserved D5 and the RRKK motifs, which are lost in the scrambled peptide. d. The colocalization efficiency of the scrambled peptide (n = 7, 0.24 ± 0.1, mean ± s.d.) was significantly decreased in comparison to FtsNcytoHis (n = 5, 0.62 ± 0.1) and FtsNcytoD5N-RAAKHis (n = 3, 0.55 ± 0.06) (P value = 1.15 × 10-4 and 1.19 × 10-3 respectively). Each dot corresponds to an independent experiment acquired within the same batch of proteins. The boxes indicate the 25–75th percentiles, whiskers the outliers, the midline indicates the median and square indicates the mean. P values were calculated using a two-tailed Student’s t-test for parametric distributions. e. Structure of the FtsA dimer in complex with the C-terminal peptide of FtsZ46. The surface charge of one FtsA monomer is shown. The 1 C domain (dashed square) contains a large negatively charged patch, which is oriented towards the membrane surface, where it can interact directly with positively charged FtsN peptide.

Source data

Extended Data Fig. 3 Differential imaging of protein patterns.

a. Top: Representative pre-processed micrographs showing the pattern of FtsZ and FtsNcytoHis as well as FtsA and FtsNcytoHis. Middle: Illustration of image subtraction process to obtain a differential image. Bottom: Fluorescent spots corresponding to growing and shrinking ends of FtsZ filament bundles and associated proteins. Micrographs show representative results from n = 16 independent experiments with FtsZ/FtsN and n = 3 for FtsA/FtsN. Scale bars are 5 µm. b. Fluorescence intensity histograms for spots of FtsZ, FtsA and FtsNcytoHis from one representative experiment. The histograms for growth and shrinkage showed similar distributions and intensities, suggesting symmetric behavior with a small shift towards brighter values for the shrinking ends. c. Differential image of fluorescent spots overlaid with result from detection (purple circles) and tracking (coloured lines). Representative image from more more than 20 independent experiments. d. Instantaneous velocities of fluorescent spots (growth) of FtsNcytoHis and FtsQcytoHis, FtsZ and FtsA and lognormal fits. FtsZ (v = 42.6 ± 32.1 nm/s, mean ± s.d.; n = 16; cyan), FtsA (v = 50.1 ± 33.5 nm/s; n = 3; green, p-value = 0.10), FtsQcytoHis (v = 46.8 ± 37.1 nm/s; n = 3; dark yellow, p-value = 0.21) and FtsNcytoHis (v = 44.3 ± 38.2 nm/s; n = 13, magenta, p-value = 0.94). P values were calculated using a two-tailed Student’s t-test for parametric distributions. e. Kymographs obtained from raw and differential movies of FtsZ, FtsA, FtsNcytoHis and FtsQcytoHis, illustrating that image subtraction simplifies detection of the dynamic behavior, representative kymographs from n = 20 rings. f. Mean-squared displacement calculated for individual trajectories. Positive slope corresponds to directed motion, red straight line illustrates diffusive behavior. g. Comparison between different methods for treadmilling speed quantification: directly from kymographs (Kymo, n = 20 from 3 (FtsA) and 5 (FtsN and FtsZ) independent experiments) and from automated treadmilling analysis via differential imaging (MSD, n(FtsZ) = 16 independent experiments with 30398 tracks total, n(FtsN) = 13 independent experiments with 20809 tracks total, n(FtsA) = 3 independent experiments with 3964 tracks total). The boxes indicate the 25–75th percentiles, whiskers the outliers, the midline indicates the median and square indicates the mean.

Source data

Extended Data Fig. 4 GMPCPP slows down filament reorganization dynamics, but does not affect colocalization between FtsZ and FtsA.

a. Filament patterns formed by FtsZ and FtsA in the presence of either GTP (top, n = 3) or GMPCPP (bottom, n = 2) before and after FtsNcytoHis addition. Scale bars are 10 µm. b. Reorganization dynamics of the FtsZ filament pattern is slowed down in the presence of GMPCPP compared to pattern formed with GTP. Graph shows temporal autocorrelation of fluorescence time lapse movies, fast decay indicates rapid reorganization. n = 3 (GTP), n = 2 (GMPCPP). Line represents the mean, error bars the standard deviation. c. PCC quantification reveals comparable colocalization between FtsZ and FtsA in the presence of different nucleotides. The presence of FtsN decreases PCC value, however this decrease is nucleotide-independent (n = 3 (GTP), n = 2 (GMPCPP)). The boxes indicate the 25–75th percentiles, whiskers the outliers, the midline indicates the median and square indicates the mean.

Source data

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.

Source data

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

a. Schematic illustration of the reconstitution approach: i. FtsNcyto-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 FtsNcyto-TM-Cy5 with Alexa Fluor 488-FtsZ. An FtsNcyto-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 FtsNcyto-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 FtsNcyto-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 FtsNcyto-TM (n = 2, N = 2) diffuses about 2-fold slower than FtsNcytoHis (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 FtsNcyto-His and 0.34 ± 0.21 µm2/s for FtsNcyto-TM without FtsA/FtsZ; 0.15 ± 0.04 µm2/s for FtsNcyto-His and 0.21 ± 0.07 µm2/s for FtsNcyto-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 FtsNcyto-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 FtsNcyto-TM (0.66 ± 0.1 s, mean ± s.d., n = 1, N = 3) shows similar confinement time as FtsNcyto-His.

Source data

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

a. The FtsZ filament pattern and colocalization with FtsNcytoHis 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 FtsNcytoHis, n = 3 (bulk diffusion constant obtained from FRAP profile analysis): Dcoef (FtsNcytoHis0%Chol) = 1.37 ± 0.31 µm2/s, Dcoef (FtsNcytoHis30%Chol) = 0.90 ± 0.21 µm2/s. After addition of FtsA and FtsZ, FtsNcytoHis shows the same diffusion constant on high and low fluidity membranes as diffusion is limited due to interaction with FtsZ-FtsA cofilaments. Dcoef (FtsNcytoHis0%Chol) = 0.18 ± 0.12 µm2/s, Dcoef (FtsNcytoHis30%Chol) = 0.10 ± 0.03 µm2/s; mean ± s.d. c. A decrease in membrane fluidity does not affect colocalization efficiency between FtsNcytoHis 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 s.d. 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.

Source data

Extended Data Fig. 8 Peptide sequences.

Sequences of peptides used in this study.

Source data

Extended Data Fig. 9

Summary of PCC values for different peptides (corresponds to Fig. 1g–h).

Extended Data Fig. 10

Summary of the binding constants for different FtsN constructs to FtsA.

Supplementary information

Reporting Summary

Supplementary Video 1

Membrane-bound FtsNcytoHis is sorted by cofilaments of FtsA and FtsZ. FtsNcytoHis is homogeneously distributed on a membrane when no other proteins are present (0–30 s; magenta; right). After the addition of FtsA and FtsZ to the reaction chamber (noisy signal in both channels), FtsZ filaments assemble on the membrane (cyan; left). Within 5 min after the addition, FtsNcytoHis is sorted into a pattern resembling the FtsZ filaments. The experiment was performed on 1 mol% mono-Ni-NTA membranes with 1.5 µM Cy5-FtsZ, 0.5 µM FtsA and 1 µM CF488-FtsNcytoHis. The video was acquired at 2 s per frame and corresponds to the micrographs in Fig. 1b (n = 5).

Supplementary Video 2

Membrane-bound FtsQcytoHis is sorted by cofilaments of FtsA and FtsZ. FtsQcytoHis is homogeneously distributed on a membrane before the addition of FtsA and FtsZ (0–1 min; yellow; middle). After the addition of FtsA and FtsZ, FtsZ filaments assemble on the membrane (cyan; left). Within several minutes, membrane-bound FtsQcytoHis is organized into a pattern resembling FtsZ filaments. The experiment was performed on 0.2 mol% tris-Ni-NTA membranes with 1.5 µM Alexa Fluor 488-FtsZ, 0.5 µM FtsA and 1 µM Cy5-FtsQcytoHis. The acquisition rate was 2 s per frame. The video corresponds to the micrographs in Fig. 1c.

Supplementary Video 3

FtsNcytoHis and FtsQcytoH is are simultaneously sorted by cofilaments of FtsA and FtsZ. FtsNcytoHis and FtsQcytoHis are simultaneously bound and homogeneously distributed on the membrane before the addition of FtsA–FtsZ (0–1 min; cyan and yellow, respectively). After the addition of FtsA and FtsZ, FtsNcytoHis and to FtsQcytoHis peptides colocalize in filament-like patterns. The experiment was performed on 0.5 mol% tris-Ni-NTA membranes with 0.15 µM Alexa Fluor 488-FtsNcytoHis, 0.75 µM Cy5-FtsQcytoHis, 0.5 µM FtsA and 1.5 µM FtsZ. The video was acquired at 2 s per frame and corresponds to Fig. 1d.

Supplementary Video 4

Colocalization of FtsN to FtsZ is mediated by FtsA. FtsNcytoHis shows similar colocalization to FtsZ (left) and FtsA (right). Top: CF488-FtsNcytoHis (magenta; middle) was added to Cy5-FtsZ-FtsA cofilaments at the steady state (cyan; left). A merge of both channels shows colocalization between CF488-FtsNcytoHis and treadmilling filaments of FtsZ (bottom left). Bottom: CF488-FtsNcytoHis (magenta; top right) was added to cofilaments of FtsZ and TMR-FtsA at steady state (green; top left). A merge of both channels shows co-migration of CF488-FtsNcytoHis and TMR-FtsA (bottom right). The experiments were acquired at 2 s per frame and correspond to the data in Fig. 1e–g.

Supplementary Video 5

Supplementary Video 5: Differential imaging of treadmilling dynamics. Top left: overlay of FtsZ and FtsNcytoHis patterns. Bottom left: overlay of the differential image of FtsZ and FtsNcytoHis. The differential video shows close coupling between FtsNcytoHis and the directional motion of treadmilling FtsZ filaments. Right: overlay of FtsNcytoHis (cyan) and FtsZ (magenta). Moving spots correspond to growing filament bundles moving along the filaments. The experiment was performed on a 1 mol% mono-Ni-NTA membrane with 0.5 µM FtsA, 1.5 µM Cy5-FtsZ and 1 µM CF488-FtsNcytoHis and was acquired at 2 s per frame. The differential video was created as described in the Methods. The video corresponds to Extended Data Fig. 3a.

Supplementary Video 6

Colocalization of FtsNcytoHis to FtsZ–FtsA filaments and the influence of treadmilling dynamics. CF488-FtsNcytoHis shows stronger colocalization to FtsZ in the presence of GTP (top) than in the presence of GMPCPP (bottom). Top: CF488-FtsNcytoHis (magenta; right) was added to the reaction chamber with FtsA and FtsZ cofilaments at steady state (cyan; left). Bottom: CF488-FtsNcytoHis (magenta; right) was added to a reaction chamber containing 0.5 µM FtsA and 1.5 µM Cy5-FtsZ (cyan; left). In both experiments, 0.5 µM FtsA, 1.5 µM Cy5-FtsZ and 1 µM CF488-FtsNcytoHis, as well as 4 mM GTP and 2 mM GMPCPP, respectively, were used. For the GTP experiments, the acquisition rate was kept at 2 s per frame, while the rate was decreased in the presence of GMPCPP to 5 s per frame to limit photobleaching. The raw files were processed to show every fifth (GTP) or every second (GMPCPP) frame, resulting in a 10 s per frame video. The videos correspond to Fig. 2c.

Supplementary Video 7

FRAP. FtsZ, FtsA and FtsNcytoHis dynamics arise from different processes, as shown by FRAP experiments on SLBs. The difference in the recovery times of the patterns is evident, with FtsZ showing the fastest recovery, mainly by exchange with FtsZ in the buffer and no influence of diffusion on the membrane, while fluorescence recovery of FtsA and FtsNcytoHis was slower and dominated by lateral diffusion. Each experiment was performed in the presence of 1.5 µM FtsZ, 0.5 µM FtsA and 1 µM FtsNcytoHis, labelled with Cy5, TMR and CF488CF, respectively. All videos were acquired at 2 s per frame and correspond to Extended Data Fig. 5b.

Supplementary Video 8

Comparison of single-molecule dynamics. Top: Single-molecule dynamics of Cy5-FtsZ, Cy5-FtsA and Cy5-FtsNcytoHis. Bottom: single-molecule dynamics merged with the corresponding Alexa Fluor 488-FtsZ pattern. Single molecules of FtsZ display no diffusive behaviour at all. Monomers within the filaments are constantly exchanged by binding/unbinding, as expected for treadmilling filaments. Single molecules of FtsA display a mixed behaviour of lateral diffusion and binding/unbinding. Single molecules of FtsNcytoHis move by fast lateral diffusion on the plane of the membrane, showing transient confinement to FtsZ–FtsA cofilaments. Each experiment was performed on 0.2 mol% tris-Ni-NTA membranes with 1.5 µM Alexa Fluor 488-FtsZ, 0.5 µM FtsA and 1 µM FtsNcytoHis present. For visualization of the single molecules, small amounts of Cy5-labelled FtsZ, FtsA or FtsNcytoHis were added (100, 75 and 75 pM, respectively). The videos were recorded at 0.228 s per frame and correspond to Fig. 3a.

Supplementary Video 9

Comparison of single-molecule trajectories of FtsNcytoHis and FtsNcyto-DDEEHis. Representative trajectories showing the differences in the diffusive motion of FtsNcyto-DDEEHis (grey track; left) and FtsNcytoHis (magenta; right). FtsNcyto-DDEEHis covers a large area and does not remain at isolated spots. FtsNcytoHis shows similar diffusive motion in the beginning until it is captured by FtsZ–FtsA cofilaments, where it shows confined behaviour. The red links represent gaps in trajectory linking.

Supplementary Video 10

Overlay of single-particle FtsNcytoHis and FtsZ pattern. Representative video of single molecules of Cy5-FtsNcytoHis in the presence of FtsA–FtsZ filaments acquired at 228 ms. Single-particle FtsN undergoes rapid random motion with transient confinement to FtsZ filaments. The video corresponds to Fig. 3f.

Supplementary Video 11

Left: diffusion of FtsNcyto-TM in a polymer-supported lipid membrane. Middle: FtsNcyto-TM shows diffusion-and-capture behaviour at normal protein concentrations (FtsA: 0.5 µM; FtsZ: 1.5 µM) and at an increased FtsZ concentration of 3 µM. Under no conditions did we find directional motion of FtsNcyto-TM.

Supplementary Tables

Supplementary Tables 1 and 2.

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Peptides physicochemical properties.

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Baranova, N., Radler, P., Hernández-Rocamora, V.M. et al. Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. Nat Microbiol 5, 407–417 (2020). https://doi.org/10.1038/s41564-019-0657-5

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