Supplemental material
JCB
THE JOURNAL OF CELL BIOLOGY
TerBush and Osteryoung http://www.jcb.org/cgi/content/full/jcb.201205114/DC1
Figure S1. Functional analysis of FtsZ C-terminal fluorescent fusion proteins. (A) Differential interference contrast micrographs of mesophyll chloroplast
phenotypes for WT Col-0, ftsZ1 knockout, and T2 ftsZ1 knockout/PFtsZ1::FtsZ1-mCerulean (T2 Comp.). (B) Graph of chloroplast numbers per cell relative to
cell area. A single plant from each genotype was analyzed. The best-fit lines have slopes of 0.0206 (R2 = 0.95), 0.0011 (R2 = 0.04), and 0.0208 (R2 =
0.88) for WT Col-0, ftsZ1 knockout, and T2 Comp., respectively. The number of mesophyll cells quantified were 73,104, and 55 for the Col-0, ftsZ1 knockout, and T2 Comp. lines, respectively. (C) Epifluorescence micrographs of FtsZ1-mCerulean signal in mesophyll cells from various leaf types from T2 Comp.
(D) Fluorescence microscopy images depicting FtsZ2-eYFP (green) assembly in an Arabidopsis chloroplast (red). Dotted line represents chloroplast outline.
(E) Differential interference contrast and fluorescence micrographs depicting FtsZ2-GFP assembly in an E. coli mutant depleted of endogenous FtsZ (E. coli
strain WM746; Ma and Margolin, 1999). In the absence of bacterial FtsZ, FtsZ2-GFP assembles filaments (arrowheads) and spots (arrows) in the bacterial
cytoplasm. Black bar, 10 µm. White bars, 5 µm.
Chloroplast FtsZ dynamics in fission yeast • TerBush and Osteryoung
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Figure S2. FtsZ1 and FtsZ2 distinct filament morphologies are independent of protein expression level. Epifluorescence micrographs of S. pombe cells expressing FtsZ1-eYFP (A), FtsZ2-eCFP (B), FtsZ1-eYFP D275A (C and D), and FtsZ2-eCFP D322A (E and F) at variable expression levels. Because the two
panels shown in each row were taken from the same image and processed identically, the difference in fluorescence intensity reflects a difference in protein
level. Cell cultures were grown in inducing conditions for 36–40 h and imaged for eYFP (FtsZ1) and eCFP (FtsZ2). Dotted lines represent cell outlines. Bars,
5 µm.
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Figure S3. Relative FtsZ1 and FtsZ2 protein levels from bulk culture extracts. Immunoblot analysis of FtsZ1-eYFP and FtsZ2-eCFP protein levels from a soluble bulk culture extract from a yeast strain coexpressing these two proteins. The blot was probed with a monoclonal GFP. The resulting band intensities,
corresponding to FtsZ1-eYFP (bottom arrow, 66 kD) and FtsZ2-eCFP (top arrow, 72 kD), were quantified, the FtsZ1-eYFP signal was normalized to 1,
and the FtsZ2-eCFP signal was normalized relative to the FtsZ1-eYFP signal. The bottom band (asterisk) likely represents a breakdown product formed during extraction, as diffuse fluorescence signal was not evident during imaging. Approximate molecular masses in kD are shown to the left.
Chloroplast FtsZ dynamics in fission yeast • TerBush and Osteryoung
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Figure S4. FtsZ1 and FtsZ2 dynamics were not altered over the range of protein levels analyzed during FRAP analysis. Graphs show individual half-time
and percent recovery values versus photomultiplier tube (PMT) voltage settings for each of the ten cells analyzed by FRAP in all four single-expression stains.
With all other microscope acquisition settings remaining constant for all experiments, PMT voltage inversely correlates to fluorescence intensity (http://
www.olympusmicro.com/primer/techniques/confocal/pmtintro.html) and therefore with protein content (high voltage = lower protein and vice versa),
though the relationship is not necessarily linear. Left y-axis represents recovery half-time (blue bars); right y-axis represents percent recovery (red bars). (A)
FtsZ1, (B) FtsZ2, (C) FtsZ1 D275A, and (D) FtsZ2 D322A expressed alone.
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Video 1. Fluorescence recovery of FtsZ1 after photobleaching. Time-lapse video of fluorescence recovery for FtsZ1 expressed
alone in S. pombe. Images were acquired on a laser-scanning confocal microscope (FluoView 1000; Olympus) and given false
color to represent fluorescence intensity (blue = weak signal; red = strong signal). Frames were collected every 3.5 s. Stills
represented in Fig. 3 A. Arrowhead indicates location of bleach. Bar, 2 µm.
Video 2. Fluorescence recovery of FtsZ2 after photobleaching. Time-lapse video of fluorescence recovery for FtsZ2 expressed
alone in S. pombe. Images were acquired on a laser-scanning confocal microscope (FluoView 1000; Olympus) and given
false color to represent fluorescence intensity (blue = weak signal; red = strong signal). Frames were collected every 10 s. Stills
represented in Fig. 3 B. Arrowhead indicates location of bleach. Bar, 2 µm.
Video 3. Fluorescence recovery of FtsZ1 D275A after photobleaching. Time-lapse video of fluorescence recovery for FtsZ1
D275A expressed alone in S. pombe. Images were acquired on a laser-scanning confocal microscope (FluoView 1000; Olympus) and given false color to represent fluorescence intensity (blue = weak signal; red = strong signal). Frames were collected
every 5 s. Stills represented in Fig. 4 A. Arrowhead indicates location of bleach. Bar, 2 µm.
Video 4. Fluorescence recovery of FtsZ2 D322A after photobleaching. Time-lapse video of fluorescence recovery for FtsZ2
D322A expressed alone in S. pombe. Images were acquired on a laser-scanning confocal microscope (FluoView 1000; Olympus) and given false color to represent fluorescence intensity (blue = weak signal; red = strong signal). Frames were collected
every 10 s. Stills represented in Fig. 4 B. Arrowhead indicates location of bleach. Bar, 2 µm.
Table S1. FRAP data for all single and coexpression strains
Yeast expression strain
FtsZ1
FtsZ1 D275A
FtsZ2
FtsZ2 D322A
FtsZ1 + FtsZ2
FtsZ1 D275A + FtsZ2 D322A
FtsZ1 + FtsZ2 D322A
FtsZ1 D275A + FtsZ2
t1/2 (s)
33.10
49.79
86.96
239.21
38.68
70.93
46.93
123.21
38.00
72.11
41.22
112.06
±
±
±
±
±
±
±
±
±
±
±
±
7.83
8.35a
22.08a
271.60
9.88
13.37
9.37
69.66
11.01
106.96
11.10
49.74
% recovery
71.32
53.91
31.07
12.09
73.74
74.82
56.66
19.27
46.83
7.57
50.37
30.74
±
±
±
±
±
±
±
±
±
±
±
±
11.51
11.03a
7.70a
10.25b
13.64
11.26b
10.31
12.47
10.44a
8.53
12.85
20.44
a
Statistically significant difference from the corresponding value in FtsZ1 single-expression strain (P < 0.01).
Statistically significant difference from the corresponding value in FtsZ2 single-expression strain (P < 0.01).
Time for one-half recovery (t1/2, s) and maximum percent recovery for each protein in each expression/coexpression strain. Data represent an average of 10 FRAP
measurements ± SD.
b
Reference
Ma, X., and W. Margolin. 1999. Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ. J. Bacteriol. 181:7531–7544.
Chloroplast FtsZ dynamics in fission yeast • TerBush and Osteryoung
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