MitochondrialMitochondrial stressstress signalingsignaling-- reactivereactive oxygenoxygen speciesspecies as as signalingsignaling moleculesmolecules
2007–2013 m. Žmogiškųjų išteklių plėtros veiksmų programos 3 prioriteto „Tyrėjų gebėjimų stiprinimas“ VP1-3.1-ŠMM-05-K priemonės „MTTP tematinių tinklų, asociacijų veiklos stiprinimas“projektas „Lietuvos Biochemikų draugijos potencialo kurti žinių visuomenę didinimas“
(NR. VP1-3.1-ŠMM-05-K-01-022)
Laboratory of Bioenergetics and Biomembranes,Nencki Institute of Experimental Biology,
Polish Academy of Sciences
Dominika Malińska
Role of mitochondria in the cell
O2.-
H2O2
[Ca2+]cADP / ATP
∆∆∆∆ΨΨΨΨH+
Ca2+
ATP
Krebs cycle
Mitochondria in regulation of cytosolic calcium fluxes
- ATP supply for Ca2+ pumps in PM and ER
- special contact sites between mitochondrial membranes and PM or ER
Role of mitochondria in the cell
O2.-
H2O2
[Ca2+]cADP / ATP
∆∆∆∆ΨΨΨΨ
Ca2+-dependent signaling
redox signaling
oxidative stress
Ca2+
ATP
Krebs cycle
cell death
Regulation of mitochondrial function
ADP / ATP
∆∆∆∆ΨΨΨΨ
- ATP demand of the cell, intensity of ATP-consuming processes
- substrate supply
ATP
Krebs cycle
[Ca2+]cADP / ATP
∆∆∆∆ΨΨΨΨ
Influence of mitochondrial calcium uptake on respiratory chain functioning
Regulation of mitochondrial function
Ca2+
ATP
Krebs cycle
O2.-
H2O2
Mitochondrial retrograde signaling - feedback information to nucleus about functional state of mitochondria
Cellular response to mitochondrial stress
Regulation of mitochondrial function
Ca2+
O2
ATP
Krebs cycle
[Ca2+]cADP / ATP
∆∆∆∆ΨΨΨΨ
O2.-
Mitochondrial retrograde signalingcell nucleus
calcineurin A,CaMKAMPK
PGC1αNRF2NRF1
HIF1α
SIRT1
succ
inat
e,
oxo
-glu
tara
te
H2O2
↑ expression of:- respiratory chain
Ca2+
O2
ATP
Krebs cycle
[Ca2+]c
succ
inat
e,
2-o
xo
ADP / ATP↑ glycolysis,
↓OxPhos
- respiratory chainproteins- enzymes related to fatty acid oxidation- mitochondrialantioxidant defenceproteins (SOD2, mGPx)
∆∆∆∆ΨΨΨΨ
Cellular response to mitochondrial dysfunction
Osteosarcoma cells
- wild type
- cybrid cells with 98% NARP mutation (ATP synthase, subunit A6)
- rho0 cells (mitoDNA depletion)
http://www.columbiamitodiagnostics.org
Cellular response to mitochondrial dysfunction
Osteosarcoma cells
- wild type
- cybrid cells with 98% NARP mutation (ATP synthase, subunit A6)
- rho0 cells (mitoDNA depletion)
WT NARP Rho0
NARP Rho0
∆ψ
[Ca2+]c
[O2.-]m
↓
Cellular response to mitochondrial dysfunction
- mitochondrial membrane potential
Flu
ores
cenc
e [%
]
[O2.-]c
Szczepanowska et al. (2004) FEBS Lett. 578: 316-322
NARP Rho0
∆ψ
[Ca2+]c
[O2.-]m
↑
↓
↑↑
Cellular response to mitochondrial dysfunction
WT NARP Rho0
+thapsigargin
(sel
ectiv
e in
hibi
tor
of C
a2+
AT
Pas
e in
the
ER
mem
bran
e)
- cytosolic calcium levels
[O2.-]c
Flu
ores
cenc
e [%
]
+thapsigargin
(sel
ectiv
e in
hibi
tor
of C
aA
TP
ase
in th
e E
R m
embr
ane)
Szczepanowska et al. [unpublished observations]
NARP Rho0
∆ψ
[Ca2+]c
[O2.-]m
↑
↑
↓
↑↑
↓
Cellular response to mitochondrial dysfunction
- intracellular superoxide concentration
[O2.-]c ↑
Wojewoda et al. (2010) Biochim Biophys Acta 1797: 890-896
Cellular response to mitochondrial dysfunction
O2.-
ADP / ATP
mitochondrial dysfunction → changes in intracellular levels of
ATP, ROS and Ca2+ → compensatory response of the cell
cellular dysfunctionprevention of cell injury
Ca2+
O2
ATP
Krebs cycle
[Ca2+]cADP / ATP
∆∆∆∆ΨΨΨΨ
ROS synthesis in mitochondria
oxidative stress
cell death
redox signaling
proliferation
differentiation
cytoprotection
ROS
ROS synthesis in mitochondria
EXPERIMENTAL METHODS:
skel
etal
mu
scle
m
ito
cho
nd
ria
ROS synthesis in mitochondria
- comparison of different tissues
bra
in
mit
och
on
dri
ask
elet
al m
usc
le
mit
och
on
dri
a
Malinska et al. (2009) Meth Enz 456: 419-437
proliferating C2C12 myoblasts
mouse skeletal muscle homogenates
ROS synthesis in mitochondria
- changes during cellular differentiation process
C2C12 cells differentiated to myotubes
brain mitochondria redox titration of CoQ
- mouse and rat brain SMP
ROS synthesis in mitochondria
III
III IVQ
succ anti Amalon
Malinska et al. (2010) Biochim Biophys Acta 1797: 1163-1170
Influence of Ca2+ on mitochondrial ROS synthesis
brain mitochondria
+
-CaU
Ca2+
III
III IVQ
succ anti A
Ru 360
Malinska et al. (2010) Biochim Biophys Acta 1797: 1163-1170
brain mitochondria
ROS generation membrane potential
Influence of Ca2+ on mitochondrial ROS synthesis
+
-CaU
Ca2+
III
III IVQ
succ anti A
Ru 360
brain mitochondria
ROS generation membrane potential
Influence of Ca2+ on mitochondrial ROS synthesis
- Ru 360 + Ru 360
succ (RET to complex I)
succ + anti A(complex III)
∆Ψ∆Ψ∆Ψ∆Ψ
TTFB
brain mitochondria
Influence of Ca2+ on mitochondrial ROS synthesis
- Ru 360 + Ru 360
Ca2+
succ (RET to complex I)
succ + anti A(complex III)
∆Ψ∆Ψ∆Ψ∆Ψ
Influence of Ca2+ on mitochondrial ROS synthesis
complex I- and complex III- dependent superoxide synthesis differ in:
- sensitivity to changes in ∆pH and ∆ψ
- intracellular compartment to which they release O2.-
- dependence on respiratory chain redox state
Summary
∆ΨΨΨΨATP
synthesisrespiratory
chain
mitochondrialCa2+ uptake
mitochondrialROS synthesis
Krebs cycle
cytosolicCa2+ levels
intracellularATP/ ADP
intracellularROS
cell functioning retrograde signaling
Acknowledgements
Nencki Institute of Experimental Biology:
Laboratory of Bioenergetics and Biomembranes - prof. Jerzy Duszyński
Laboratory of Intracellular Ion Channels - prof. Adam Szewczyk
Bonn University
Department of Epileptology – prof. Wolfram S. Kunz
Laboratory of Bioenergetics and Biomembranes
Jerzy Duszyński, Lech Wojtczak,Joanna Szczepanowska,Marta Wojewoda,Mariusz Więckowski,Magdalena Lebiedzińska,Jan Suski,Dominika MalińskaMałgorzata Bejtka,Violetta Biernat
research techniques:- isolation of mitochondria and mitochondria-associated membranes- analysis of respiratory chain activity in isolated organelles and in whole cells (colorimetric activity determination after Blue Native gel electrophoresis; respirometric analysis)- visualisation of intracellular structures with laser scanning cytometry and confocal microscopy- fluorescent measurements of intracellular ROS levels, Ca2+ fluxes, Ca2+ levels in different organelles, mitochondrial membrane potential