fisb text 2012-12-31 - rudner lab | harvard medical school
TRANSCRIPT
wt ΔfisB
T 2
T 2.
5 T
3 T
3.5
T 4
T 4.
5 T
1.5
phase membranes CFP membranes phase CFP
Figure S2 Doan et al.
Figure S4 Doan et al.
0
50
100 His-FisBECD His-FisBFL
A28
0nm
(mA
U)
A28
0nm
(AU
)
monomer
Elution volume (mL)
0
50
100
150
200 66
13.6
Elution volume (mL)
monomer
oligomer oligomer
Figure S6 Doan et al.
-IPTG
+IP
TG
S10
0
FT
P10
0
Frx
1
Frx
2
Frx
3
Frx
4
His-FisB
M -IPTG
+IP
TG
S10
0
FT
P10
0
Frx
1
Frx
2
Frx
3
Frx
4
mock
M
anti-HIS
Frx
1 Fr
x 2
Frx
3 Fr
x 4
Frx
1 Fr
x 2
Frx
3
Frx
4
mock His-FisB
Frx
1
Frx
2
Frx
3
Frx
4
Frx
1
Frx
2
Frx
3
Frx
4
mock His-FisB B
A
Figure S8 Doan et al.
YFP
-Fis
B
mem
bran
es
mer
ge
wt spoIIIE36 ΔspoIIIE B
FisB
σF
wt
2 2.5
3 3.5
4
spoIIIE36 ΔspoIIIE
2 2.5
3 3.5
4 2 2.5
3 3.5
4 Δfis
B
1.5
1.5
1.5
A
Figure S11 Doan et al.
wt ∆fisB ∆sigG ∆fisB ∆sigG ∆fisB ∆sigK
mem
bran
es
phas
e m
erge
3.8% 30.9% 4.3% 4.2% 27.8% % lysis:
1
SUPPLEMENTAL MATERIAL
SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Cells lacking SpoIIIE have defects in membrane migration. Membranes from
a wild-type (PY79) and a SpoIIIE null mutant (BDR1066) were visualized with TMA-
DPH at hour 2 (top) and 2.5 (bottom) of sporulation. Examples of sporulating cells in
which membrane migration is impaired are highlighted (yellow carets). The images are
from cells mounted on polylysine-treated coverslips (A) or on agarose pads containing
resuspension medium (B).
Figure S2. Cells lacking FisB are specifically impaired in membrane fission. Membrane
migration and fission were monitored by fluorescence microscopy during a sporulation
time course. Cells were imaged every 30 min from hour 1.5 to 4.5 in wild-type (BKM15)
and a FisB mutant (BTD3200). Both strains contained a fluorescent forespore reporter
(PspoIIQ-cfp) to detect all forespores. The membranes (false-colored red) were visualized
with FM4-64 and merged with the CFP signal (false-colored green). Yellow carets
Examples of sporulating cells that had undergone membrane fission are highlighted
(yellow carets). Examples of cell lysis are indicated (red carets).
Figure S3. Comparison of the membrane dyes FM4-64 and TMA-DPH. Wild-type
sporulating cells (BKM15) at hour 3 of sporulation were stained with FM4-64 and TMA-
DPH. This strain contained a fluorescent forespore reporter (PspoIIQ-cfp) to detect all
forespores. Examples of sporulating cells that had undergone membrane fission are
highlighted (yellow carets). In these cells the forespore membranes are not detectable by
FM4-64 but are weakly stained by TMA-DPH.
Figure S4. FisB forms high molecular weight oligomers in vitro. Detergent-solubilized
full-length His-tagged FisB (His-FisBFL) and soluble His-tagged FisB extracellular
domain (His-FisBECD) were purified by affinity chromatography on Ni2+-NTA agarose
and then analyzed by size-exclusion chromatography on a Superdex 200 10/300 GL
column. All fractions were analyzed by immunoblot (not shown). Monomer and
2
oligomers are indicated. In both cases, the proteins were soluble after centrifugation at
100,000Xg. For His-FisBECD, the elution volumes for three standard proteins (amylase,
200 kDa; BSA, 66 kDa; cytochrome C, 13.6 kDa) are indicated (red carets).
Figure S5. Low-level expression of YFP-FisB (strain BTD3124) reveals specific
localization at the cell pole at the time of fission. Larger fields of cells showing the
localization of YFP-FisB by fluorescence microscopy during a time course of sporulation
from hour 2, 2.5 and 3. The membranes from the same fields were visualized with TMA-
DPH (false-colored red) and merged with the YFP-FisB signal (false-colored green).
White carets (at hour 2 of sporulation) highlight examples of the punctate YFP-FisB
localization pattern prior to fission. Yellow carets show examples of sporulating cells
discrete YFP-FisB foci at or near the cell pole.
Figure S6. Purified full-length His-FisB (His-FisB) and mock-purified proteins.
Purifications were from E. coli cells harboring the His6-FisBfull-length expression plasmid
(pDT355) and the empty expression plasmid (pET28a). A complete description of the
purifications can be found in Text S1. (A) Fractions from the protein purification were
analyzed by SDS-PAGE and coomassie blue staining. The red arrow indicates the
monomeric form of His-FisB and the red bracket highlights SDS-dependent His-FisB
multimers. (B) The eluted fractions from the Ni-NTA column (from separate
purifications) were analyzed by immunoblot using anti-His antibodies. The red arrow
indicates the monomeric form of His-FisB and the red bracket highlights SDS-dependent
His-FisB multimers. The nature of the SDS-induced aggregation of His-FisB is not
known but is reminiscent of SDS-induced aggregation of neuronal SNARES (Hayashi et
al. 1994).
Figure S7. SNARE proteins were purified and reconstituted into liposomes as described
in (Weber et al. 1998) except lipid-to-protein ratios were decreased to 1:200 for Vamp2
and 1:400 for t-SNAREs. The liposome fusion assay was performed as described in
Figure 4, except the temperature was increased to 37˚C. As a control, t-SNARE
liposomes were incubated with the cytoplasmic domain of Vamp2 (cdv) in excess to
3
block fusion. After 2 hours, detergent was added to determine the maximum
fluorescence. The data was normalized and plotted as a percent of maximum fluorescence
versus time in minutes.
Figure S8. Mutations in spoIIIE affect FisB levels and/or localization. (A) Immunoblot
analysis of whole cell lysates from sporulating cells. FisB levels were analyzed in
sporulating cells from wild-type (strain PY79), a FisB mutant (BDR1083), spoIIIE36
(BDR1050) and a SpoIIIE null mutant (BDR1066) using anti-FisB antibodies. σF levels
were monitored to control for efficiency of sporulation and loading. Time (in hours) after
the initiation of sporulation is indicated. (B) YFP-FisB localization was monitored by
fluorescence microscopy at hour 3 of sporulation in wild-type (BTD3124), a spoIIIE36
mutant (BTD3143), and a SpoIIIE null mutant (BTD3142). The membranes were
visualized using TMA-DPH (false-colored red) and merged with the YFP-FisB signal
(false-colored green). Examples of sporulating cells in which YFP-FisB forms foci at the
cell pole are highlighted (yellow carets).
Figure S9. Engulfment membrane fission and septal membrane fission (cytokinesis) have
opposite membrane topologies. Schematic representations showing membrane fission
during asymmetric division (top) and at the end of engulfment (bottom). A single-pass
membrane protein (with a green ball in the cytoplasm) is shown to highlight the opposite
topologies.
Figure S10. σG is activated in the absence of membrane fission. σG activity was
monitored in single cells using a σG-responsive promoter fusion to cfp (PsspB-cfp) in wild-
type (BTD1609) and a FisB mutant (BTD2045). Cells were visualized at hour 3.5 of
sporulation. The membranes (false-colored red) were visualized with FM4-64 and
merged with the CFP signal (false-colored green). Examples of sporulating cells in which
the forespores have σG activity in the absence of membrane fission (yellow carets) or
after membrane fission has occurred (blue carets) are indicated. White carets highlight
sporulating cells that have not completed membrane fission in which σG activity is not
detected in the forespore.
4
Figure S11. Lysis in the FisB mutant is suppressed in the absence of σG. Membrane
fission (monitored with FM4-64 and PspoIIQ-cfp) and cell lysis (assessed by phase
contrast) were analyzed 5 hours after the onset of sporulation in wild-type (BKM15), a
FisB mutant (BTD3200), a sigmaG mutant (ΔsigG, BTD3218), a FisB, sigG double
mutant (BTD3219), and a FisB, sigmaK double mutant (ΔfisB, ΔsigK, BTD3217). For
each strain, the percentage of lysed cells compared to total sporulating cells (including
lysed cells) is indicated below the image. A subset of cells lacking FisB lyse at this time
point. Lysis is suppressed in the absence of the forespore transcription factor σG but not
in the absence of mother cell transcription factor σK. This result suggests that σG-
dependent gene expression in cells that have not undergone membrane fission causes cell
lysis. Yellow carets highlight examples of lysed cells.
Movie M1A and B. GFP-FisB forms dynamic foci in the mother-cell membranes. GFP-
FisB (strain, BAM003) localization was monitored during sporulation at hour 2.5 by
time-lapse fluorescence microscopy. The focal plane was at the bottom surface of the
cell. Images were acquired every 5 seconds.
Movie M2. GFP-FisB foci are immobile at the cell pole at the time of membrane fission.
GFP-FisB (strain, BAM003) localization was monitored during sporulation at hour 2.5 by
time-lapse fluorescence microscopy. The focal plane was at mid-cell. Images were
acquired every 30 seconds.
SUPPLEMENTAL TEXT
Protein purification
His6-FisBFL and His6-FisBECD fusion proteins were expressed in E. coli BL21 DE3 pLysS
and purified by affinity chromatography on Ni2+-NTA agarose (Qiagen). MBP-FisBECD
fusion protein was expressed in E. coli NB42 and purified by affinity chromatography on
an amylose resin (New England Biolabs).
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For His6-FisBECD, cells were grown in LB with 20 µg/ml kanamycin at 16˚C to an OD600
of 0.6 and induced overnight at 16˚C by the addition of IPTG to 1 mM. Cells were
harvested by centrifugation and resuspended in 1/50th volume buffer I (50 mM Tris-HCl
pH 8, 200 mM NaCl, 5 mM 2-Mercaptoethanol, 1 mM PMSF, and 10 mM imidazole)
and flash-frozen in N2(l). A crude extract was prepared by freeze-thawing the cells
followed by two passes through a French press. A soluble fraction from 100,000Xg spin
was loaded on a 1 mL Ni2+-NTA agarose (Qiagen) column equilibrated with buffer I.
Bound protein was washed with buffer II (20 mM Tris-HCl pH 8, 200 mM NaCl, 5 mM
2-Mercaptoethanol, 2% glycerol, 10 mM imidazole) and eluted in buffer III (20 mM Tris-
HCl pH 8, 200 mM NaCl, 5 mM 2-Mercaptoethanol, and 150 mM imidazole).
For His6-FisBFL (and mock-purified proteins) cells were grown in LB with 20 µg/ml
kanamycin at 30˚C to an OD600 of 0.6 and induced by the addition of IPTG to 1 mM.
After 3 h of induction, cells were harvested by centrifugation and resuspended in 1/50th
volume buffer I. A crude extract was prepared by freeze-thawing the cells followed by
two passes through a French press. The membrane fraction was isolated by 100,000Xg
spin and resuspended in 1/250th volume buffer B (20 mM Tris-HCl pH 8, 200 mM NaCl,
2% glycerol, 5 mM 2-Mercaptoethanol, 1 mM PMSF, and 10 mM imidazole). Membrane
proteins were solubilized by the addition of the nonionic lipid-like detergent foscholine-
12 (Anatrace) to a final concentration of 2%. The mixture was rotated at 4˚C for 2 hours.
Soluble and insoluble fractions were separated by centrifugation at 100,000Xg for 1 hour
at 4˚C. The soluble fraction was collected and loaded on a Ni2+-NTA agarose (Qiagen)
column equilibrated with buffer I containing 0.1% foscholine-12. Bound protein was
washed with buffer II containing 0.1% foscholine-12 and eluted in buffer III containing
0.1% foscholine-12.
For MBP-FisBECD, cells were grown in LB with 100 µg/ml ampicillin at 37˚C to an
OD600 of 0.6 and induced by the addition of IPTG to 1 mM. After 2 h of induction, cells
were harvested by centrifugation and resuspended in 1/50th volume buffer A (20 mM
Tris-HCl pH 8, 1 M NaCl, 1 mM EDTA, 5 mM 2-Mercaptoethanol, 1 mM PMSF) and
flash-frozen in N2(l). A crude extract was prepared by freeze-thawing the cells followed
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by two lysis cycles in a French press. A soluble fraction was made by 100,000Xg spin
and was loaded on 1ml of amylose resin (New England Biolabs) equilibrated with buffer
A. Bound protein was washed with buffer A and eluted in buffer A containing 10 mM
maltose.
Plasmid construction
pKM008 [amyE::PspoIIQ-cfp (spec)] was generated in a two-way ligation with a HindIII-
BamHI PCR fragment containing the cfp gene (codon optimized for B. subtilis; (Doan et
al. 2005)) with an optimized RBS and pKM001 cut with HindIII and BamHI. pKM001
[amyE::PspoIIQ (spec)] was built in a two-way ligation with an EcoRI-HindIII PCR
fragment containing the spoIIQ promoter (oligonucleotide primers oDR234 and oDR235
and template DNA pDR172 [amyE::PspoIIQ-gfp (kan)] (DZR, unpublished)) and pLD30
(Garsin et al. 1998) cut with EcoRI and HindIII.
pDT343 [ycgO::PspoIID-RBSfisB-yfp-fisB (erm)] was built in a three-way ligation with a
HindIII-XhoI PCR fragment containing the coding sequence of YFP with a modified fisB
RBS (oligonucleotide primers oTD269 and oDR108 and plasmid template pKL183
(Lemon and Grossman 2000), a XhoI-BamHI PCR product containing the FisB coding
sequence (oligonucleotide primers oDR328 and oDR329 and PY79 genomic DNA as
template) and pDT92 [ycgO::PspoIID-spoIVB (erm)] (TD and DZR, unpublished) cut with
HindIII and BamHI.
pDT357 [ycgO::fisB (erm)] was generated in a two-way ligation with a NheI-BamHI
PCR fragment containing the fisB locus (oligonucleotide primers oTD291 and oTD118
and PY79 genomic DNA as template) and pKM84 cut with NheI and BamHI. pKM84 [ycgO::erm] is an ectopic integration vector for double crossover insertions into the nonessential ycgO locus (KM and DZR, unpublished).
pDT240 [mbp-fisBECD] was generated in a two-way ligation with a BamHI-PstI PCR
product encoding the FisB extracellular domain (216 amino acids) (oligonucleotide
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primers oTD234 and oTD235 and PY79 genomic DNA as template) and pMAL-C2x
(New England Biolabs) cut with BamHI and PstI.
pDT355 [his6-fisBfull-length] was built in a two-way ligation with a NheI-XhoI PCR
fragment encoding full length FisB (oligonucleotide primers oTD279 and oDR406 and
PY79 genomic DNA as template) and pET28a (Novagen) cut with NheI and XhoI.
pDT389 [his6-fisBECD] was generated in a two-way ligation with a BamHI-XhoI PCR
product encoding the FisB extracellular domain (216 amino acids) (oligonucleotide
primers oTD306 and oTD307 and pDT355 as template) and pRsetA (Invitrogen) cut with
BamHI and XhoI.
pAM002 [ycgO::PfisB-gfp-fisB (cat)] was generated in a three-way ligation with a
HindIII-XhoI PCR product containing monomeric gfp preceded by the native fisB RBS
and the fisB TTG start codon (oligonucleotide primers oDR834 and oDR108), a XhoI-
BamHI PCR product containing the fisB coding sequence (oligonucleotide primers
oDR835 and oDR836), and pAM001 cut with HindIII and BamHI. pAM001 [ycgO::PfisB
(cat)] was built in a two-way ligation with an EcoRI-HindIII PCR product
consisting of 300 nucleotides preceding the fisB coding sequence (oligonucleotide
primers oDR832 and oDR833) and pKM077 (ycgO::cat) cut with EcoRI and
HindIII. pKM077 [ycgO::cat] is an ectopic integration vector for double crossover insertions into the nonessential ycgO locus (KM and DZR unpublished).
Supplemental Reference
Doan T, Marquis KA, Rudner DZ. 2005. Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum. Mol Microbiol 55: 1767-1781.
Garsin DA, Paskowitz DM, Duncan L, Losick R. 1998. Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor sigmaF in Bacillus subtilis. J Mol Biol 284: 557-568.
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Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Südhof TC, Niemann H. 1994. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J 13: 5051-5061.
Lemon KP, Grossman AD. 2000. Movement of replicating DNA through a stationary replisome. Mol Cell 6: 1321-1330. Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Söllner TH,
Rothman JE. 1998. SNAREpins: minimal machinery for membrane fusion. Cell 92: 759-772.
Table 1. Strains used in this study
Strain Genotype Source PY79 Prototrophic wild-type strain Youngman et al., 1983 BDR1050 spoIIIE36 Wu and Errington, 1994 BDR1066 spoIIIE::neo Wu and Errington, 1994 BDR1083 fisB::tet Eichenberger et al., 2002 BKM15 amyE::PspoIIQ-cfp (spec) This work BTD1609 yycR::PsspB-cfp (phleo) Doan et al., 2009 BTD2045 yycR::PsspB-cfp (phleo), fisB::tet This work BAM027 yycR::PspoIIQ-cfp (phleo), dynA::cat This work BTD3124 fisB::tet, ycgO::PspoIID-RBSfisB-yfp-fisB (erm) This work BTD3142 fisB::tet, ycgO::PspoIID-RBSfisB-yfp-fisB (erm), spoIIIE::neo This work BTD3143 fisB::tet, ycgO::PspoIID-RBSfisB-yfp-fisB (erm), spoIIIE36 This work BTD3200 amyE::PspoIIQ-cfp (spec), fisB::tet This work BTD3217 amyE::PspoIIQ-cfp (spec), fisB::tet, spoIVCB::erm This work BTD3218 amyE::PspoIIQ-cfp (spec), spoIIIG::neo This work BTD3219 amyE::PspoIIQ-cfp (spec), fisB::tet, spoIIIG::neo This work BAM003 fisB::tet ycgO::PfisB-gfp-fisB (cat) This work
Table 2. Plasmids used in this study
plasmid description source pKM008 amyE::PspoIIQ-cfp (spec) This work pDT343 ycgO::PspoIID-RBSfisB-yfp-fisB (erm) This work pDT357 ycgO::fisB (erm) This work pDT240 mbp-fisBECD This work pDT355 his6-fisBfull-length This work pDT389 his6-fisBECD This work pAM002 ycgO::PfisB-gfp-fisB (cat) This work
Table 3. Oligonucleotide primers used in this study
primer sequence oDR108 cggCTCGAGtttgtatagttcatccatgc oDR234 gccGAATTCcatgcttcgtcaatgtatatgctg oDR235 cggAAGCTTagcaacattctgaacacttttctg oDR328 cggCTCGAGttgccaagatatcgcggccc oDR329 gccGGATCCcgcatttgagggcgtgg oDR406 cggCTCGAGccgcctcttcaacaggcg oTD118 gccGGATCCttatttgctgcttttttctttttt oTD234 gttGGATCCggctcgatcaaacctgtttta oTD235 cggCTGCAGttatttgctgcttttttcttttt oTD269 gttAAGCTTatgtaaggggggattatgagtaaaggagaagaactt oTD279 gccGCTAGCttgccaagatatcgcggcc oTD291 ggcGCTAGCggccgtcgccaatgatcac oTD306 gtgCTCGAGttatttgctgcttttttctttttt oTD307 cgcGGATCCaatggctcgatcaaacctgt oDR832 gccGAATTCccaaaacgaattttatggcc oDR833 cgcAAGCTTcatatcacaatgtatgcttgtaag oDR834 gcgAAGCTTaaggggggattttcttgagtaaaggagaagaacttttc oDR835 cggCTCGAGttgccaagatatcgcggcc oDR836 cgcGGATCCcgcatttgagggcgtg oAM028 ctgtgcaggaggagccagcggac oAM029 agggtaactattgccgtatgctgtcataagtctgaacccc oAM030 ggggttcagacttatgacagcatacggcaatagttaccct oAM031 gccaactgaattttacattttggagctgtaatataaaaaccttc oAM032 gaaggtttttatattacagctccaaaatgtaaaattcagttggc oAM033 gcgttcagcttttcaagcatacgctc
capital letters indicate the endonuclease recognition sequences