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Dissecting the distinct functions of mitochondria
4th International Conference and Exhibition on Metabolomics & Systems Biology
April 29, 2015 – Philadelphia, USA
Inmaculada Martinez-Reyes – Dr. Navdeep Chandel Lab – Northwestern University, Chicago
Mitochondrial metabolism is necessary
for tumorigenesis.
Mitochondria as signaling organelles.
Glucose
Pyruvate
AMP/ATP
Catabolism
AMPK
AcetylCoA
ATP-Citrate
Lyase
OAA
TCA
Cycle
H+
V
Acetyl-CoA
Citrate
α-KG
α-KG
ADP
HAT
Me
NAD+
ATP
e-
O2
IV
H+
NADH
FADH2
H2O
e-
e- e-
c
c
FAD
e-
III
H+
Q
Q
e-
II
JMJD3
I
Q
H+
NFkB
HIF
H2O2
?
Ac
What functions of mitochondrial
metabolism are necessary for
cell proliferation and epigenetics?
Two functions of mitochondria: TCA cycle metabolites and
membrane potential.
H+
ETC
NADH/FADH2
TCA
Metabolites
(Acetyl-CoA, Succinyl-CoA)
O2
H2O
H+
NAD+/FAD
ADP
ATP
H+
Y
(ATP, ROS, Iron-sulfur clusters)
DNA polymerase gamma (POLG) is necessary for
mitochondrial DNA (mtDNA) replication.
Dominant Negative (DNPOLG)
Doxycycline
Dominant Negative (DNPOLG)
Doxycycline
Doxycycline (DOX)
Hans Spelbrink
Dominant negative POLG depletes mtDNA
encoded RNA transcripts.
WT PLOG + DOX
Day 9
DN PLOG + DOX
Day 0
Day 3
Day 6
Day 9
Ty Wang
Janine Santos
Dominant negative POLG depletes mtDNA
encoded proteins.
WT-POLG
0
-
3
-
6
-
9
-
0 3
+ +
DN-POLG
6
+
9
+
Days
DOX
0
-
3
-
6 9 0
- +
3 6 9
+ + +
Days
DOX
COXII
COXII
SDHA
SDHA
Tubulin
Tubulin
Loss of mitochondrial DNA does not
induce cell death.
W T -P O L G
100
D N -P O L G
% v ia b ility
80
60
40
20
0
D ay 0
D ay 3
D ay 6
D ay 9
-
+
+
+
DOX
1 .5
t o D a y 0 ( In t a c t c e lls )
R e la t iv e M ito c h o n d r ia l O C R
Loss of mitochondrial DNA decreases oxygen
consumption rate (OCR).
W T -P O L G
D N -P O L G
1 .0
0 .5
0 .0
D ay 0
D ay 3
D ay 6
D ay 9
-
+
+
+
DOX
( N o r m a liz e d T M R E - C C C P )
M e m b r a n e P o te n tia l
Loss of mitochondrial DNA decreases mitochondrial
membrane potential.
1 .5
1 .0
0 .5
W T -P O L G
D N -P O L G
0 .0
D ay 0
D ay 3
D ay 6
D ay 9
-
+
+
+
DOX
Loss of mitochondrial DNA increases
glucose catabolism.
D N -P O L G
10
( A r b it r a r y U n it s )
D y e R e d u c t io n
D ay 0
D ay 3
8
D ay 6
6
D ay 9
4
2
0
a -D -G lu c o s e
Biolog
Loss of mitochondrial DNA induces dependence on
glycolysis for survival.
D e a d c e lls ( % P I+ )
100
D N -P O L G
80
60
40
20
0
+
-
-
+
Day 0
-
+
-
+
-
+
-
D ay 3
+
+
+
-
-
+
D ay 6
D ay 9
+
+
G lu c o s e
G a la c to s e
DOX
Loss of mitochondrial DNA induces AMPK
activation.
DN-POLG
0
-
0
-
3
3
6
6
9
9
Days
+
+
+
+
+
+
DOX
p-AMPK
AMPK
Tubulin
310
5
310
6
210
5
210
6
210
5
210
6
110
5
110
6
510
4
510
5
W T -P O L G
D N -P O L G
C e ll n u m b e r
C e ll n u m b e r
Loss of mitochondrial DNA diminishes cell
proliferation rate.
W T -P O L G
D N -P O L G
0
0
+
+
+
DOX
1 .5  1 0
7
1 .0  1 0
7
W T -P O L G
5 .0  1 0
D N -P O L G
6
C e ll n u m b e r
C e ll n u m b e r
7
DOX
Day 6
Day 3
2 .0  1 0
+
210
8
110
8
510
7
W T -P O L G
D N -P O L G
0
0
+
+
Day 9
DOX
+
+
Day 12
DOX
Quantification of Histone modifications using Silac.
D N -P O L G
( N o r m a liz e d t o d a y 0 )
D ay 0
D ay 3
D ay 6
1 .0
0 .5
c
7
9
a
c
6
K
3
H
H
3
K
5
3
2
K
H
3
K
3
H
a
c
a
c
2
7
a
c
a
8
1
K
3
H
H
3
3
K
K
1
9
4
a
a
c
c
0 .0
H
R e la tiv e e x p r e s s io n
1 .5
He Huang
Minimal changes in methylation and acetylation of H2B and H4.
Yingming Zhao
Loss of mitochondrial DNA decreases specific
histone H3 acetylation.
DN-POLG
0
-
3
+
6
+
9
+
Days
DOX
H3K9ac
H3K14ac
H3K18ac
H3K27ac
H3 total
Mitochondria metabolism is necessary for
cell proliferation and specific histone
acetylation.
Two functions of mitochondria: TCA cycle metabolites and
membrane potential.
H+
ETC
NADH/FADH2
TCA
Metabolites
(Acetyl-CoA, Succinyl-CoA)
O2
H2O
H+
NAD+/FAD
ADP
ATP
H+
Y
(ATP, ROS, Iron-sulfur clusters)
Electron transport chain couples electron flux to
proton pumping.
H+
H+
H+
Cyt c
C-I
C-II
NADH
NAD+
C-IV
Q
FADH2
FAD
C-III
O2
H2O
NDI1 mimics complex I and AOX bypasses
complex III and complex IV.
H+
H+
H+
Cyt c
Q
C-I
NADH
NAD+
C-III
C-IV
NDI1
NADH
NAD+
C-II
FADH2
FAD
AOX
O2
O2
H2O
H2O
Eric Dufor
NDI1 and AOX expression uncouples electron flux from
proton pumping in cells with depleted mitochondrial DNA.
Q
NDI1
C-II
NADH
NAD+
FADH2
FAD
AOX
O2
H2O
AOX-NDI1 expression restores electron flux but not
membrane potential.
NDI/
AOX
NADH/FADH2
TCA
Metabolites
O2
ATP4-
H2O
ADP3-
NAD+/FAD
Y
AOX-NDI expression does not restore
mitochondrial DNA.
C O X II/S D H A D N A r a t io
1 .5
D N - P O L G - A O X /N D I1
1 .0
0 .5
0 .0
D ay 0
-
D ay 3
+
D ay 6
+
D ay 9
+
DOX
G F P /B F P D a y 0
600
A O X /N D I1 D a y 0
400
200
0
0
10
20
T im e ( m in )
30
P yr + M al
SHAM
Rot + Ant
300
G F P /B F P D a y 6
P e r m e a b iliz e d C e ll s
SHAM
Rot + Ant
O x y g e n C o n s u m p t io n R a te (p m o l/m in )
P yr + M al
P e r m e a b iliz e d C e ll s
O x y g e n C o n s u m p t io n R a te (p m o l/m in )
AOX-NDI expression restores oxygen consumption
rate in isolated mitochondria.
A O X /N D I1 D a y 6
200
100
0
0
10
20
30
T im e ( m in )
Seahorse Biosciences
Study of the metabolic profile of the cells
-2.00
2.00
DN-POLG-GFP/BFP Day 0
DN-POLG-GFP/BFP Day 3
DN-POLG-GFP/BFP Day 6
DN-POLG-GFP/BFP Day 9
DN-POLG-AOX/NDI1 Day 0
DN-POLG-AOX/NDI1 Day 3
DN-POLG-AOX/NDI1 Day 6
DN-POLG-AOX/NDI1 Day 9
Amino Acid
Carbohydrate
Cofactors and Vitamins
Energy
Lipid
Nucleotide
Peptide
Xenobiotics
Study of the metabolic profile of the cells
Super Pathw ay
Sub Pathw ay
Senescent 3d
Senescent 0d
Senescent 6d
Senescent 0d
Senescent 9d
Senescent 0d
Active 3d
Active 0d
Active 6d
Active 0d
Active 9d
Active 0d
0.875
1
1.25
1.5
2
Alanine and Aspartate Metabolism
Glutamate Metabolism
Methionine, Cysteine, SAM and Taurine Metabolism
Urea cycle; Arginine and Proline Metabolism
Creatine Metabolism
Glycolysis, Gluconeogenesis, and Pyruvate Metabolism
Carbohydrate
Pentose Phosphate Pathw ay
Pentose Metabolism
Nucleotide Sugar
Aminosugar Metabolism
Energy
TCA Cycle
Oxidative Phosphorylation
Long Chain Fatty Acid
Polyunsaturated Fatty Acid (n3 and n6)
Fatty Acid Metabolism
Glycerolipid Metabolism
Purine Metabolism, (Hypo)Xanthine/Inosine containing
Purine Metabolism, Adenine containing
Purine Metabolism, Guanine containing
Pyrimidine Metabolism, Orotate containing
Nucleotide
Pyrimidine Metabolism, Uracil containing
Pyrimidine Metabolism, Cytidine containing
Pyrimidine Metabolism, Thymine containing
Purine and Pyrimidine Metabolism
Nicotinate and Nicotinamide Metabolism
Riboflavin Metabolism
Cofactors and VitaminsPantothenate and CoA Metabolism
Biotin Metabolism
Folate Metabolism
Tetrahydrobiopterin Metabolism
Pterin Metabolism
Fold of Change
0.75
AOX-NDI expression maintains levels of TCA cycle
metabolites.
1 .5
F u m a r a te
C itr a te
A b u n d a n c e (A .U )
2 .0
1 .5
1 .0
A b u n d a n c e (A .U )
2 .5
1 .0
0 .5
0 .5
0 .0
0 .0
Day 0
Day 3
Day 6
Day 9
Day 0
Day 3
Day 6
Day 0
Day 9
1 .0
0 .5
A b u n d a n c e (A .U )
A lp h a -K e to g lu ta r a te
M a la te
Day 9
Day 0
Day 3
Day 6
Day 9
D N - P O L G A O X N D I1
4
1 .5
A b u n d a n c e (A .U )
Day 6
D N -P O L G
D N - P O L G A O X N D I1
D N -P O L G
Day 3
3
2
1
0 .0
Day 0
Day 3
Day 6
Day 9
Day 0
Day 3
Day 6
Day 9
0
Day 0
D N -P O L G
Day 3
Day 6
Day 9
Day 0
Day 3
Day 6
D N - P O L G A O X N D I1
D N -P O L G
D N - P O L G A O X N D I1
Day 9
( N o r m a liz e d T M R E - C C C P )
M e m b r a n e P o te n tia l
AOX-NDI does NOT restore mitochondrial
membrane potential.
1 .5
D N - P O L G G F P /B F P
D N - P O L G A O X /N D I1
1 .0
0 .5
0 .0
D ay 0
-
D ay 3
D ay 6
D ay 9
+
+
+
DOX
AOX-NDI1 expression restores electron flux (TCA
cycle) but not membrane potential.
NDI/
AOX
NADH/FADH2
TCA
Metabolites
O2
ATP4-
H2O
ADP3-
NAD+/FAD
Y
Is electron flux (TCA cycle) sufficient to
restore cell proliferation and histone
acetylation?
Electron flux (TCA cycle) is sufficient to restore
histone acetylation.
DN-POLG-AOX/NDI1
3
/H
3
c
/H
a
7
a
2
8
K
3
H
9
K
3
H
K
3
H
c
c
a
c
a
7
2
8
1
K
3
H
/H
3
/H
3
a
c
a
4
1
K
3
H
H3 total
c
/H
/H
3
3
/H
c
a
9
K
3
0 .0
1
H3K27ac
0 .0
0 .5
K
H3K18ac
3
0 .5
1 .0
H
H3K14ac
3
1 .0
/H
H3K9ac
D ay 9
1 .5
c
D ay 9
D ay 0
a
D ay 0
4
DOX
1
+
+
K
+
3
-
D N - P O L G - A O X /N D I1
2 .0
H
Days
3
9
( N o r m a liz e d t o d a y 0 )
3D N - P O6L G
R e la tiv e e x p r e s s io n
0
1 .5
H
( N o r m a liz e d t o d a y 0 )
R e la tiv e e x p r e s s io n
2 .0
Electron flux (TCA cycle) is NOT sufficient
to restore cell proliferation.
Electron flux (TCA cycle) is NOT sufficient to
restore increased glucose catabolism.
D N - P O L G - A O X N D I1
D N - P O L G - G F P /B F P
10
10
D ay 0
6
4
2
( A r b it r a r y U n it s )
D ay 6
8
D y e R e d u c t io n
( A r b it r a r y U n it s )
D y e R e d u c t io n
D ay 0
D ay 6
8
6
4
2
0
0
a -D -G lu c o s e
a -D -G lu c o s e
Electron flux (TCA cycle) is NOT sufficient to restore
glucose dependency for survival.
D N - P O L G - A O X - N D I1
D e a d c e lls ( % P I+ )
100
80
60
40
20
0
+
-
-
+
+
-
+
-
+
-
+
+
-
+
Day 0
D ay 3
D ay 6
D ay 9
-
+
+
+
G lu c o s e
G a la c to s e
DOX
Electron flux without proton pumping is NOT sufficient
to alleviate energetic stress.
DN-POLG-AOX/NDI1
0
-
0
-
3
3
6
6
9
9
+
+
+
+
+
+
Days
DOX
p-AMPK
AMPK
Actin
Two functions of mitochondria: TCA cycle metabolites and
membrane potential.
H+
ETC
NADH/FADH2
TCA
Histone Acetylation
O2
H2O
H+
NAD+/FAD
ADP
ATP
H+
? Y
Cell Proliferation
Chandel Lab
Nav Chandel
Sam Weinberg
Heywon Kong
Lauren Diebold
James Eisenbart
Manan Metha
Colleen Reczek
Arianne Rodriguez
Michael Schieber
Acknowledgements
Collaborators
He Huang
Yingming Zhao
Ty Wang
Janine Santos
Eric Dufor
Hans Spelbrink
Ralph Deberardinis
Loss of ATPIF1 allows the maintenance of mitochondrial membrane potential
Loss of mitochondrial DNA decreased mitochondrial
mass.
( N o r m a liz e d t o d a y 0 )
M ito c h o n d r ia l M a s s
1 .5
1 .0
0 .5
W T -P O L G
D N -P O L G
0 .0
D ay 0
D ay 3
D ay 6
D ay 9
-
+
+
+
DOX
Loss of mitochondrial DNA alters mitochondrial morphology
A b u n d a n c e (A .U )
A c e ty l-C o A
2 .0
1 .5
1 .0
0 .5
0 .0
Day 0
Day 3
D N -P O L G
Day 6
Day 9
Day 0
Day 3
Day 6
D N - P O L G A O X N D I1
Day 9
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