Dominika Malińska. Laboratory of Bioenergetics and Biomembranes, Nencki Institute of Experimental Biology, Polish Academy of Sciences
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1 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 ) Mitochondrial stress signaling - reactive oxygen species as signaling molecules Dominika Malińska Laboratory of Bioenergetics and Biomembranes, Nencki Institute of Experimental Biology, Polish Academy of Sciences
2 Role of mitochondria in the cell H 2 O 2 O 2.- ADP / ATP [Ca 2+ ] c H + Ψ ATP Krebs cycle Ca 2+
3 Mitochondria in regulation of cytosolic calcium fluxes - ATP supply for Ca 2+ pumps in PM and ER - special contact sites between mitochondrial membranes and PM or ER
4 redox signaling Role of mitochondria in the cell H 2 O 2 Ca 2+ -dependent signaling oxidative stress O 2.- ADP / ATP [Ca 2+ ] c Ψ ATP cell death Krebs cycle Ca 2+
5 Regulation of mitochondrial function - ATP demand of the cell, intensity of ATP-consuming processes - substrate supply ADP / ATP Ψ ATP Krebs cycle
6 Regulation of mitochondrial function Influence of mitochondrial calcium uptake on respiratory chain functioning ADP / ATP [Ca 2+ ] c Ψ ATP Krebs cycle Ca 2+
7 Regulation of mitochondrial function Mitochondrial retrograde signaling - feedback information to nucleus about functional state of mitochondria Cellular response to mitochondrial stress H 2 O 2 O.- 2 ADP / ATP [Ca 2+ ] c Ψ ATP Krebs cycle Ca 2+
8 Mitochondrial retrograde signaling cell nucleus HIF1α NRF1 PGC1α NRF2 glycolysis, OxPhos su uccinate, 2-ox xo-glutarate H 2 O 2 O.- 2 Ψ SIRT1 AMPK ADP / ATP calcineurin A, CaMK [Ca 2+ ] c expression of: - respiratory chain proteins - enzymes related to fatty acid oxidation - mitochondrial antioxidant defence proteins (SOD2, mgpx) ATP Krebs cycle Ca 2+
9 Cellular response to mitochondrial dysfunction Osteosarcoma cells - wild type - cybrid cells with 98% NARP mutation (ATP synthase, subunit A6) - rho0 cells (mitodna depletion)
10 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
![- ] m [O 2.](/docs-images/84/89502952/images/11-2.jpg "- ] c Fluorescence [%]")
11 Cellular response to mitochondrial dysfunction - mitochondrial membrane potential NARP Rho0 ψ [Ca 2+ ] c [O 2.- ] m [O 2.- ] c Fluorescence [%] Szczepanowska et al. (2004) FEBS Lett. 578:
![[Ca 2+ ] c [O 2.- ] m [O 2.](/docs-images/84/89502952/images/12-2.jpg "- ] c Fluorescence [%]")
12 Cellular response to mitochondrial dysfunction - cytosolic calcium levels NARP Rho0 ψ WT NARP Rho0 [Ca 2+ ] c [O 2.- ] m [O 2.- ] c Fluorescence [%] +thapsigargin (selective inhibitor of Ca 2+ ATPase in the ER memb brane) Szczepanowska et al. [unpublished observations]
![Rho0 ψ [Ca 2+ ] c](/docs-images/84/89502952/images/13-4.jpg "[O 2.- ] m [O 2.")
![- ] c Wojewoda et](/docs-images/84/89502952/images/13-5.jpg "al. (2010) Biochim")
13 Cellular response to mitochondrial dysfunction - intracellular superoxide concentration NARP Rho0 ψ [Ca 2+ ] c [O 2.- ] m [O 2.- ] c Wojewoda et al. (2010) Biochim Biophys Acta 1797:
14 Cellular response to mitochondrial dysfunction mitochondrial dysfunction changes in intracellular levels of ATP, ROS and Ca 2+ compensatory response of the cell prevention of cell injury cellular dysfunction O.- 2 ADP / ATP [Ca 2+ ] c Ψ ATP Krebs cycle Ca 2+
15 ROS synthesis in mitochondria ROS oxidative stress redox signaling cell death cytoprotection differentiation proliferation
16 EXPERIMENTAL METHODS: ROS synthesis in mitochondria
17 - comparison of different tissues ROS synthesis in mitochondria brain mitochondria sk keletal muscle mitochondria Malinska et al. (2009) Meth Enz 456:
18 ROS synthesis in mitochondria proliferating C2C12 myoblasts - changes during cellular differentiation process mouse skeletal muscle homogenates C2C12 cells differentiated to myotubes
19 ROS synthesis in mitochondria brain mitochondria redox titration of CoQ - mouse and rat brain SMP I II Q III IV malon succ anti A Malinska et al. (2010) Biochim Biophys Acta 1797:
20 Influence of Ca 2+ on mitochondrial ROS synthesis brain mitochondria Ru 360 CaU I II Q III IV + - Ca 2+ succ anti A Malinska et al. (2010) Biochim Biophys Acta 1797:
21 Influence of Ca 2+ on mitochondrial ROS synthesis brain mitochondria ROS generation membrane potential Ru 360 CaU I II Q III IV + - Ca 2+ succ anti A
22 Influence of Ca 2+ on mitochondrial ROS synthesis brain mitochondria ROS generation membrane potential succ (RET to complex I) succ + anti A (complex III) Ψ - Ru Ru 360
23 Influence of Ca 2+ on mitochondrial ROS synthesis brain mitochondria TTFB succ (RET to complex I) succ + anti A (complex III) Ψ Ca 2+ - Ru Ru 360
24 Influence of Ca 2+ 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 O dependence on respiratory chain redox state
25 Summary respiratory chain Ψ ATP synthesis Krebs cycle mitochondrial ROS synthesis mitochondrial Ca 2+ uptake intracellular ROS cytosolic Ca 2+ levels intracellular ATP/ ADP cell functioning retrograde signaling
26 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
27 Laboratory of Bioenergetics and Biomembranes Jerzy Duszyński, Lech Wojtczak, Joanna Szczepanowska, Marta Wojewoda, Mariusz Więckowski, Magdalena Lebiedzińska, Jan Suski, Dominika Malińska Małgorzata 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, Ca 2+ fluxes, Ca 2+ levels in different organelles, mitochondrial membrane potential
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