c (ii) (iii) (iv) (tail pcr) - nature...

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DOI: 10.1038/ncb1907

Figure S1 Generation of Drp1-/- cells and mice. a, Map of Drp1-targeting constructs. Wild-type Drp1 locus (Drp1+), targeted Drp1 locus (Drp1lox), and knockout Drp1 locus (Drp1-). The arrowheads indicate the loxP sequence.

The arrows indicate primers used for genotyping by PCR. b, Schematic diagram of constructions. c, Identification of floxed, or targeted Drp1 by PCR genotyping.

a

c

bES Drp1+/+ mouse Drp1lox/+

transfection: targeting vector selection in low G418 x mouse EIIa-Cre x mouse Nestin-CreES Drp1lox/+ mouse Drp1lox/lox mouse Drp1lox/+; EIIa-Cre mouse Drp1lox/+; Nestin-Cre selection in high G418 backcross x mouse Drp1lox/lox

ES Drp1lox/lox MEF Drp1lox/lox mouse Drp1+/- mouse Drp1lox/lox; Nestin-Cre (iv) Adeno-Cre Adeno-Cre intercrossES Drp1-/- (i) MEF Drp1-/- (ii) mouse Drp1-/- (iii)

(ii) (iii) (iv)(tail PCR)

Drp1lox allele

XbaI

Drp1+ allele

SpeI

3.6kb 1.8kb 5.6kb

EcoRV2 3 4 5 6HindIII

Drp1- allele

1.9kb2 3 4 5 6

3 4 5 6

neo

0.2

0.40.6

(kbp) MW

5

2

1

Adeno - LacZ Cre

-

+/+ lox/lox lox/+lox/lox lox/lox

(i)

lox

+

+

lox/lox -/-

-

lox

+

+ +

+/+ +/- -/-

flox+

-

flox/+ flox/flox; nes-Cre#1 #2

flox

+

Cre

(tail PCR)

Figure S1. Generation of Drp1-/- cells and mice. a, Map ofDrp1-targeting constructs. Wild-type Drp1 locus (Drp1+),targeted Drp1 locus (Drp1lox), and knockout Drp1 locus(Drp1-). The arrowheads indicate the loxP sequence. The

arrows indicate primers used for genotyping by PCR. b,Schematic diagram of constructions. c Identification of floxed, or targeted Drp1by PCR genotyping.

© 2009 Macmillan Publishers Limited. All rights reserved.


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Figure S2 a, Morphology of the endoplasmic reticulum (GFP-cytochrome b5) and Golgi (YFP-Golgi) in controls and Drp1-/- MEFs was analyzed by fluorescence microscopy. Mitochondria (mit) were visualized by su9-RFP. b and c, MEFs and ES cells that had been transfected with or without the expression vector for rat Drp1 were treated with or without

20 µM CCCP for 2 h, then mitochondrial morphology was analyzed as in (a). Mitochondria with the indicated morphology in CCCP-treated or untreated MEFs and ES cells were quantified. At least 100 cells in 3 distinct fields were analyzed. d, Representative images in (b) are shown.

a

dlox/lox lox/lox CCCP -/- -/- CCCP

MEF

MEFlox/lox

mit/ER

-/-

mit/ER

lox/lox

mit/Golgi

-/-

mit/Golgi

b c

0

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100

lox/lox lox/lox CCCP

-/- -/- CCCP

tubularintermediatefragment

ESce

ll co

unt (

%)

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100

lox/lox CCCP

lox/lox+Drp1CCCP

-/- CCCP

-/- +Drp1CCCP

highly connectedtubularfragment

MEF

cell

coun

t (%

)

Figure S2. a, Morphology of the endoplasmic reticulum(GFP-cytochrome b5) and Golgi (YFP-Golgi) in controls andDrp1-/- MEFs was analyzed by fluorescence microscopy.Mitochondria (mit) were visualized by su9-RFP. b and c,MEFs and ES cells that had been transfected with or withoutthe expression vector for rat Drp1 were treated with or

without 20 µM CCCP for 2 h, then mitochondrialmorphology was analyzed as in A. Mitochondria with theindicated morphology in CCCP-treated or untreated MEFsand ES cells were quantified. At least 100 cells in 3 distinctfields were analyzed. d, Representative images in (b) areshown.




Figure S3 a, Analysis of the Ψ levels across the mitochondrial inner membrane. Control and Drp1-/- MEFs were cultured in the presence of 20 nM MitoTracker Red or 100 nM MitoTracker Green for 1 h. The fluorescent signal in each cell was counted for more than 100 cells in 3 distinct optical fields. b, Respiration of MEFs through complex I, II and IV. The harvested cells were incubated with the indicated respiratory substrates, and O2 consumption was measured using oxygen electrode. c, MEFs were incubated with or without 10 µM oligomycin for 10h and the cell extracts

were subjected to ATP concentration analysis using luciferase. d, Wild-type and Drp1-/- MEFs were cultured in amino acids- and serum-depleted medium for 1 h in the presence or absence of lysosomal protease inhibitors (E64d + Pepstatin A). The cell lysates were subjected to SDS-PAGE and subsequent immunoblotting using the antibodies against LC3 and a-tubulin. e, Wild-type and Drp1-/- MEFs were cultured as in (d) for the indicated time periods, incubated with the antibodies against LC3 and Tom70, and analyzed by immunofluorescence microscopy.

Figure S3. a, Analysis of the Ψ levels across themitochondrial inner membrane. Control and Drp1-/- MEFswere cultured in the presence of 20 nM MitoTracker Red or100 nM MitoTracker Green for 1 h. The fluorescent signal ineach cell was counted for more than 100 cells in 3 distinctoptical fields. b, Respiration of MEFs through complex I,II and IV. The harvested cells were incubated with the indicated respiratory substrates, and O2 consumption was measured using oxygen electrode. c, MEFs were incubated with or without 10 M oligomycin for 10h and the cell

extracts were subjected to ATP concentration analysis using luciferase. d, Wild-type and Drp1-/- MEFs were cultured in amino acids- and serum-depleted medium for 1 h in the presence or absence of lysosomal protease inhibitors (E64d + Pepstatin A). The cell lysateswere subjected to SDS-PAGE and subsequent immunoblottingusing the antibodies against LC3 and -tubulin. e, Wild-type and Drp1-/- MEFs were cutured as in (d) for the indicated time periods, incubated with the antibodies against LC3 and Tom70, and analyzed by immunofluorescence microscopy.

a b levels Respiration c ATP levels

complex I complex II complex IV0

20

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glutamate/malate succinate/rotenone TMPD/ascorbate/antimycin

O2 c

onsu

mpt

ion

(% o

f con

trol)

lox/lox -/-

0

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red green

starvationE64d+PepA

0 1 1 0 1 1

LC3 III

-tubulin

- +- - +-

lox/lox -/-

mit LC3lox/lox

10 mm

-/-

starv 2h starv 6h

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P (%

of c

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-18kD-16kD




Figure S4 Time-lapse imaging of mitotic Drp1-/- MEFs expressing su9-RFP and YFP-α-tubulin. b, Mitochondria and a mitotic spindle in living cells were observed by fluorescence video microscopy for 1 h at 37°C at 2-min intervals. a, Representative images from (b) were depicted.

10 m

a

b

5 m

MEF -/-

MEF -/-

Figure S4. Time-lapse imaging of mitotic Drp1-/- MEFexpressing su9-RFP and YFP-α-tubulin. b, Mitochondria anda mitotic spindle in living cells were observed by fluorescence

video microscopy for 1 h at 37°C at 2-min intervals. a,Representative images from (b) were depicted.




Figure S5 Response of Drp1-/- ES cells and MEFs to proapoptotic reagents. a, ES cells were treated with 1 µM staurosporine (STS) or 20 µM actinomycin D (ActD) and the processed PARP was quantified by immunoblotting as in Fig. 3b setting the amount of unprocessed PARP at 100%. b, ES cells were treated with proapoptotic reagents for the indicated periods and the cells with the cytochrome c in the cytosol were quantified by immunofluorescence microscopy. At least 100 cells in more than 3 distinct fields were analyzed. c, Drp1-/- MEFs were treated with etoposide for the indicated periods, and the cells were analyzed by immunofluorescence microscopy. The cells with released Smac/Diablo and cytochrome c were counted. At least 100 cells in

3 independent experiments were counted. d, The cells obtained in (c) were analyzed by SDS-PAGE and subsequent immunoblotting using antibodies against the caspase 3 fragment and Tom40 as a loading control. e, MEFs were treated with actinomycin D for the indicated periods and exposure of phosphatidylserine on the outer plasma membrane was measured by flow cytometry after staining the cells with annexin V-FITC. f and g, Drp1-/- MEFs expressing Tim8-FLAG were treated with actinomycin D for the indicated periods and the number of cells that contained Tom8a-released but cytochrome c-unreleased mitochondria was counted. Thirty cells each from 3 distinct optical fields were analyzed. Scale bar, 20 µm.

Figure S5. Response of Drp1-/- ES cells and MEFs toproapoptotic reagents. a, ES cells were treated with 1 µMstaurosporine (STS) or 20 µM actinomycin D (ActD) and theprocessed PARP was quantified by immunoblotting as in Fig.3b setting the amount of unprocessed PARP at 100%. b, EScells were treated with proapoptotic reagents for the indicatedperiods and the cells with the cytochrome c in the cytosolwere quantified by immunofluorescence microscopy. At least100 cells in more than 3 distinct fields were analyzed. c,Drp1-/-MEFs were treated with etoposide for the indicatedperiods, and the cells were analyzed by immunofluorescencemicroscopy. The cells with released Smac/Diablo andcytochrome c were counted. At least 100 cells in 3

independent experiments were counted. d, The cells obtained in (c) were analyzed by SDS-PAGE and sub-sequent immunoblotting using antibodies against the caspase 3 fragment and Tom40 as a loading control. e, MEFs were treated with actinomycin D for the indicated periods and exposure of phosphatidylserine on the outer plasma membrane was measured by flow cytometry after staining the cells with annexin V-FITC. f and g, Drp1-/-MEFs expressing Tim8-FLAG were treated with actino-mycin D for the indicated periods and the number of cells that contained Tom8a-released but cytochrome c-unre-leased mitochondria was counted. Thirty cells each from3 distinct optical fields were analyzed. Scale bar, 20 m.

ES

cont-/-

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(100 M)

cyt.

c re

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16h 24h 16h 24h 16h 24h

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ActD (20 M ) STS (1 M )

c MEF

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cont-/-

ActD (20 M ) STS (1 M )

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PAR

P cl

eava

ge(r

elat

ive

activ

ity)

d MEF etoposide (200 M)0 h 4 h 8 h 12 h

Cleavedcas p-3

Tom40

cont -/- cont -/- cont -/- cont -/-cont -/- cont -/- cont -/- cont -/-

MEF

100 101 102 103 104

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0100 101 102 103 104100 101 102 103 104

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Cou

nts

Annexin V

12h:38.0%

8h:15.3%

0h:5.5%

-/- Act.D 0h8h

12h

Act.D 0h8h

12h

e

444

Annexin V

Cou

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100 101 102 103 10

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0100 101 102 103 10100 101 102 103 10

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Drp1-/- ME F

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Drp1+/+ Drp1-/- Drp1+/+ Drp1-/-0

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= 3

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Drp1+/+ Drp1-/- Drp1+/+ Drp1-/-0

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= 3




Figure S6 a, Coronal sections of the brain from E15.5 controls or Drp1lox/lox; nestin-Cre mice embryos were HE-stained. b, Paraffin-embedded brain sections of E18.5 Drp1lox/lox; nestin-Cre embryos were examined by

immunofluorescent microscopy using antibodies against the complex III core 1 protein. Forebrain regions are shown. Clustered mitochondria were observed in the Drp1-/- mouse brain.

1 mm

a E15.5 HE

cont

lox/lox; nestin-Cre

5 m

b

lox/

lox;

nes

tin-C

reco

nt

mit mit/DAPIE18.5

Figure S6. a, Coronal sections of the brain from E15.5controls or Drp1lox/lox; nestin-Cre mice embryos wereHE-stained. b, Paraffin-embedded brain sections of E18.5Drp1lox/lox; nestin-Cre embryos were examined by

immunofluorescent microscopy using antibodies against thecomplex III core 1 protein. Forebrain regions are shown.Clustered mitochondria were observed in the Drp1-/- mousebrain.




Figure S7 Parasagittal sections of cerebella from E20 control (A) and NS-Drp1-/- (B) mice. Remarkable overall size reduction of the cerebellum as well as the brainstem is noted in NS-Drp1-/- mouse. While the thickness of the forming cerebellar cortex (EGL+PL) and the formation of the DN in the NS-Drp1-/- cerebellum are comparable to those in the control, the

white matter of the former shows marked hypoplasia. Note the apparent enlargement of the subdural/subarachnoid space in the posterior fossa in NS-Drp1-/- mouse (asterisk). S; skull, EGL; external granular layer, PL; Purkinje cell layer, DN; dentate nucleus, asterisk: subdural/subarachnoid layer. Bar=100 µm.

Figure S7. Parasagittal sections of cerebella from E20 control (A) and NS-Drp1-/- (B) mice. Remarkable overall size reduction of the cerebellum as well as the brainstem is noted in NS-Drp1-/- mouse. While the thickness of the forming cerebellar cortex (EGL+PL) and the formation of the DN in the NS-Drp1-/- cerebellum are comparable to

those in the control, the white matter of the former shows marked hypoplasia. Note the apparent enlargement of the subdural/subarachnoid space in the posterior fossa in NS-Drp1-/- mouse (asterisk). S; skull, EGL; external granular layer, PL; Purkinje cell layer, DN; dentate nucleus, asterisk: subdural/subarachnoid layer. Bar=100 m.




Figure S8 Full scans of the Western blots from the figures.

Fig 3a

GAPDH

PARP

Tom40

Tom40

Bax

Bax

GAPDH

GAPDH

cont

-/-

0h

0h

8h

8h

Fig 3b

Fig 3fFig 2h

Fig S3d

-tubulin

LC3

Fig S5d

Drp1 OPA1 Mfn1 Mfn2

MIB-tubulincatalase

Tom22Tim44Tom40 Tim17

Tom70 mhsp70Fis1 hsp60Tom20

MEF

Figure S8. Full scans of the Western blots from the figures.


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