A zip file of all movies can be downloaded here.

 

Movie S1: FtsA and FtsZ filaments move directionally together inside and outside of the division site. Cells were imaged using TIRF microscopy at 3 second intervals. Representative 5-minute acquisitions, displayed at 15 frames per second (45x actual speed). Scale bar: 2 痠.

Column 1: mNeonGreen-FtsZ (bAB185) showing filament motion.

Column 2: FtsA-mNeonGreen(SW) (bAB167) showing filament motion.

Column 3: FtsA-HaloTag(SW)-JF549 (red) and mNeonGreen-FtsZ (green) showing filament motion. bAB229 cells were incubated with 500 nM of HaloTag-JF549 ligand for 15 minutes.

Column 4: mNeonGreen-FtsZ (bAB221) showing filament motion with FtsAZ overexpressed. Cells were grown with 100 然 IPTG for 1 hour. 

 

Movie S2: Imaging of vertically oriented cells in microholes shows FtsZ moves directionally in dense constricting Z rings.

mNeonGreen-FtsZ (strain SH41) dynamics in vertically immobilized cells with 600-1000 nm radii. Cells were imaged by epifluorescence at 1 second intervals. 2 minute acquisition displayed at 15 frames per second (15x actual speed). Scale bar: 1 痠.

 

Movie S3: Pbp2B moves directionally around AZ rings

A. JF549-HaloTag-Pbp2B (bGS31) shows Pbp2B single molecules by TIRF microscopy. Cells were incubated with 50 pM of HaloTag-JF549 ligand for 15 minutes, washed and prepared as indicated in methods. Cells were imaged at 1 second intervals using TIRF microscopy. Images of mNeonGreen-FtsZ were used to mark the position of the division machinery (first 60 frames of the movie) before imaging of JF549. Arrowheads indicate Z rings that colocalize with directionally moving Pbp2B. 4-minute acquisition displayed at 30 frames per second (30x actual speed).

B Overexpression of exogenous mNeonGreen-Pbp2B (100然 IPTG, bAB146) shows diffusion of Pbp2B molecules along the cell by TIRF microscopy. Cells were imaged at 100 millisecond intervals. Scale bar: 2 痠.

 

Movie S4: FtsAZ filament motion is not stopped by inhibition of cell wall synthesis.

FtsA-mNeonGreen(SW) (bAB167) showing filament motion during perturbations to cell wall synthesis. Antibiotics were added to the top of the pad 5 minutes before imaging, as follows: no antibiotic control (top left); 50 mg/痞 Vancomycin (top right); 10 mg/痞 Ampicillin (center left); 10 mg/痞 Penicillin G (center right); 100 mg/痞 Cephalexin (bottom left) and 100 mg/痞 Fosfomycin (bottom right). Cells were imaged at 1 second intervals with TIRF microscopy. Representative 5-minute acquisitions, displayed at 30 frames per second (30x actual speed). Scale bar: 2 痠.

 

Movie S5: FtsAZ filament motion is not dependent on Pbp2B.

A mNeonGreen-FtsZ (bAB185) shows absence of Z ring constriction following depletion of Pbp2B. Cells were grown to mid-exponential phase with 10 然 IPTG, inducer was washed out from medium and cells were imaged after 1 hour of growth without IPTG. Images were acquired at 30 second intervals for 1.5 hours using epifluorescence illumination. Movie displayed at 30 frames per second (900x actual speed).

B mNeonGreen-FtsZ (bAB185) continues to show FtsZ filament motion following depletion of Pbp2B. Cells were imaged with 2 hours of Pbp2B depletion (after no Z ring constriction was detected). Cells were imaged at 1 second intervals using TIRF microscopy. Representative 5-minute acquisitions, displayed at 30 frames per second (30x actual speed). Scale bars: 2 痠.

 

Movie S6: FtsAZ filament motion is driven by treadmilling.

A Low expression of mNeonGreen-FtsZ (bAB219) shows single molecules of FtsZ are immobile at division sites. Cells were imaged at 500 millisecond intervals for 2 minutes by TIRF microscopy.  Movie displayed at 30 frames per second (15x actual speed).

B Single molecules of FtsA-HaloTag(SW)-JF646 (bAB213) are immobile within division sites. Cells were imaged at 200 millisecond intervals for 1 minute using TIRF microscopy. Representative videos are displayed at 30 frames per second (6x actual speed). Scale bars: 2 痠.

 

Movie S7: FtsZ GTPase activity modulates FtsAZ filament treadmilling and directional motion.

A Inducible FtsA-mNeonGreen(SW)-FtsZ(D213A) (bAB217) titration showing motion of FtsA filaments in presence of different induction levels of the GTPase-defective FtsZ(D213A). Cells were grown until mid-exponential phase, diluted and incubated 1 hour with 1 然 (top left), 5 然 (top right), 10 然 (bottom left) and 100 然 IPTG (bottom right). Cells were imaged at 3 second intervals for 5 minutes with TIRF microscopy. Movie displayed at 15 frames per second (45x actual speed).

B mNeonGreen-FtsZ (bAB185) showing motion of FtsZ filaments after the addition of MciZ peptide by perfusion in a CellAsic microfluidic chamber. Cells were imaged in CH medium (top left), then 10 minutes (top right) and 20 minutes (bottom left) after the addition of 1 然 MciZ. Sequentially, MciZ was washed out from cells for 10 minutes and cells were imaged again (bottom right). Cells were imaged at 3 second intervals for 5 minutes with TIRF microscopy. The exogenous addition (or expression) of MciZ causes two phases, 1) Initially (between 0 and 10 minutes after exogenous addition of induction of MciZ), small FtsZ filaments move around the cell at a faster rate. During this period, we see that all Z rings constricted faster than usual. At later time points (from 15 to 20 minutes), as the MciZ incubation time (or expression) increased, all FtsZ filaments and Z rings in the cell disappeared and did not return. Movie displayed at 15 frames per second (45x actual speed). Scale bars: 2 痠.

 

Movie S8: PC190723 blocks FtsAZ filament and Pbp2B directional motions.

A mNeonGreen-FtsZ (bAB185) showing motion of FtsZ filaments during treatments of PC190723 added by perfusion in a CellAsic microfluidic chamber. Cells were imaged at 3 second intervals in CH medium for 10 minutes, then for 10 minutes with 10 然 PC190723 (top right) and for 20 minutes while washing out the drug. Movie displayed at 30 frames per second (90x actual speed).

B JF549-HaloTag-Pbp2B (bGS31) showing Pbp2B single molecules by TIRF microscopy during PC190723 treatment. Cells were incubated with 50 pM of HaloTag-JF549 ligand for 15 minutes prior to imaging. Cells were imaged 18 minutes after the addition of 3 痞 of 10 mg/ml PC190723 on top of a 500 痞 2% agarose pad. Snapshot images of mNeonGreen-FtsZ were used to mark the position of the division machinery (first 60 frames of the movie, white arrowheads). Cells were imaged at 1 second intervals for 4 minutes with TIRF microscopy. Movie displayed at 30 frames per second (30x actual speed). Scale bars: 2 痠.

 

Movie S9: FtsAZ(D213A) overexpression creates slow-growing FtsAZ spirals.

FtsA-mNeonGreen(SW)-FtsZ(D213A) (bAB217) showing the growth of FtsA spirals by spinning-disk confocal microscopy. Cells were grown without inducer, placed under a 2% agarose CH pad containing 100 然 IPTG, then imaged at 30 seconds intervals for 2 hours. In order to make the transition from ring to spirals visible, histogram bleach correction was applied to all time-lapses in Fiji to normalize signal between frames. Representative videos are displayed at 30 frames per second (900x actual speed). Scale bar: 2 痠.

 

Movie S10: FtsAZ filament velocity controls the rate of cytokinesis.

mNeonGreen-Pbp2B rings within cells of different genetic backgrounds were followed over time by confocal microscopy. Cells were imaged at 1 minute intervals for 2 hours. 6 examples out of 12 different conditions are shown as follows: wild type (top left), FtsAZ overexpression (top right), MciZ overexpression (center left), native FtsZ-T111A mutant (center right), 5 然 IPTG of FtsZ(D213A) mutant (bottom left) and FtsZ(D213A) (10 然 IPTG) of mutant (bottom right). Representative 30-minute videos are displayed at 8 frames per second (480x actual speed). Scale bar: 2 痠.