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.