Fluorescence Correlation Spectroscopy (FCS): application for mitochondrion investigation

1. Fluorescence Correlation Spectroscopy(FCS): application for mitochondrion investigation Irina Perevoshchikova

2. Today I am going to tell about… Measurement of membrane potential on the level of solitary particles Investigation of functional state of porin (VDAC) in outer mitochondrial membrane In isolated mitochondria by Fluorescence Correlation Spectroscopy (FCS) D. Magde et al. 1972

3. Diffusion Enzymatic Activity Phase Fluctuations Conformational Dynamics Rotational Motion Protein Folding Observation Volume In Fluorescence Correlation Spectroscopy Fluctuations are the Fluorescence Signal diffusion Example of processes that could generate fluctuations

4. Defining Observation Volume: One- & Two-Photon Excitation. 2 - Photon 1 - Photon Defined by the pinhole size, wavelength, numerical aperture of the objective Defined by the wavelength and numerical aperture of the objective

5. 40x 1,2 NA water immersion objective Dichroic mirror Nd:YAG solid state laser 532 nm Pinhole, 50 mm Avalanche photodiode A scheme of our setupon the basis of inverted Olympus microscope Trace of fluorescence Autocorrelation function PCH Photon counting histogram

6. Photon Counts time  Average Fluorescence t + t t Calculating the Autocorrelation Function t3 t4 t5 t2 t1

7. z0=2,1 mm average intensity of fluorescence average brightness per molecule - - r0=0,42 mm Vconf=2,2 fl The analytical form of the autocorrelation function 3 D diffusion, one-photon excitation td – diffusion time D – diffusion coefficient

8. <N> = 2 <N> = 4 The Effects of Particle Concentration on the Autocorrelation Curve 1\N 1/N

9. The Effects of Particle Size on the diffusion time Diffusion Constants 300 um2/s 90 um2/s 71 um2/s Slow diffusion Slow Diffusion Fast diffusion Fast Diffusion Stokes-Einstein Equation: and Monomer --> Dimer Only a change in D (hence td ) by a factor of 21/3, or 1.26

10. The Autocorrelation functions of mitochondria, liposomes and fluorescent dyes Mitochondria

11. Trace of fluorescence and Photon Counting Histogram (PCH) Trace of fluorescence 1 MHZ 50 ms Probability/bin Fluorescence intensity, kHz Stirring condition 1. Non-energized state Δφ≈0 + succinate 2. Energized state • TMRE • TMRE 0,03 μМ, mitochondria, rotenone • Addition of succinate • Addition of DNP Δφ≈-200 мВ PCH + DNP 3. Deenergized state Δφ≈0 Tetramethylrhodamine, ethyl ester, TMRE

12. 2 MHz 50 ms Influence of membrane potential on the fluorescence fluctuations of charged hydrophobic probes in different membrane systems. Submitochondrial particles, oxonol VI BacteriaBacillus subtilis, TMRE Mitochondria, Safranine O 1 – Energized state, 2 – Deenergized state 1 – Non-energized state, 2 - Energized state, 3 – Deenergized state

13. Trace of fluorescence of identical fluorescent spheres (1 mm in diameter)

14. The aim of this work was to develop an approach based on the FCS method for analysis fluorescence fluctuations induced by movements of stained mitochondria in suspension. This is a road to quantification of potential on a single mitochondrion level

15. z z0 r0 x Theoretical background Quasi-cylindricalprofile of the observation volumecan bedescribed by a Gaussian distribution of the detected intensity [Rigler R. et al. 1993]: B0 – brightness of molecule, proportional to quantum yield of fluorescence, A – maximal intensity of light in the center of the confocal volume, r - a minimal distance to axis z and a coordinate z Introducing variables x=r/r0, y=z/z0, and R=(x2+y2)1/2. Then, the maximal intensity will be: The portion of events having the amplitude more than F0(i.e., P(F>F0)) is R2/R02 , where R0 is a radius of a circle in the x,y space,where all events take place. By expressing R through F we obtain: for and for Experimentally not probability P(F>F0), but number of events with fluorescence intensity higher than the definite level N(F>F0) was measured and N(F>F0)~P(F>F0). P0 – factor which is proportional to number of particle in suspension

16. Fluorescent beads, d=1 mm N(F>F0) Gaussian distribution F0 Finitial amplitude Lorentzial distribution F0, kHz Derivative of P(F>F0) gives the number of events in some interval around F=F0 and easily might be obtained by differentiation of equation: (Gaussian distribution) In some cases of one-photon excitation profile of the observation volumecan bedescribed by a Lorentzial distribution [Hess S., Webb W., 2002]: Then function P(F>F0)and its derivative are: (Lorentzial distribution) for and for Win EDR (Gaussian-Lorentzial distribution)

17. N(F>F0) F0, кГц N(F>F0) F0, kHz Peak Intensity Analysis (PIA) Р(F>F0)- probability to detect particle with fluorescence value upper than F0 Р0- factor proportional to particle concentration in suspension А∙В0 – brightness of particle in center of confocal volume N(F>F0)~P(F>F0) Suspension of fluorescent particles • d=1 μм, А∙В0= 11700 kHz • d=0,5 μм, А∙В0=1600 kHz • d= 0,1 μм, А∙В0=1080 kHz Suspension of latex beads (d=0,8 μm) doped with different concentration of TMRE TMRE, nM • d=1 μм, <I>=11000 kHz • d=0,5 μм, <I>=5600 kHz • d= 0,1 μм, <I>=2100 kHz From autocorrelation function, N does not depend on stirring

18. Protein concentration, mg/ml Number of particles, particles/ml One particle detection condition Energized mitochondria doped with TMRE Fluorescent beadsd=0,5 μм

19. 4000 kHz N(F>F0) ms F0, kHz 1 – mitochondria, rotenone А∙В0 =1880kHz, Р0=400 2 – addition of succinate А∙В0 =6600kHz, Р0=540 3 – addition of DNP А∙В0 =2760 kHz, Р0=314 N(F>F0) F0, kHz PIA application to suspension of mitochondria doped with TMRE PCRh-PE liposomes 0,0044 mg/ml mitochondrial protein АВо = 750 кHz Sof lipid molecule= 0.7 nm2 Diameter of liposome = 80 nm (Zsizer Nano) Nrhodamine= 574 in one liposome Brightness of one rhodamine molecule = 1,3 кГц Number of TMRE molecules accumulated by one mitochondrion under energized condition Concentration of TMRE inside one mitochondrion Membrane potential value

20. 1-0 mM DNP 2-5 mM DNP 3-10 mM DNP 4-15 mM DNP 5-20 mM DNP 6-25 mM DNP 7-30 mM DNP N(F>F0) F0, kHz Effect of DNP on brightness of energized mitochondria Δφ=14 mV 30 nM TMRE, 7 mM rotenone, 7 mM succinate, 0,03 mg/ml mitochondrial protein

21. Voltage Dependent Anion Cannel (VDAC) Ujwal R. et al, 2008 voltage-dependent anion channel (VDAC) serves as a global regulator, or governor, of mitochondrial function (Lemasters and Holmuhamedov, Biochim Biophys Acta 1762:181–190, 2006) Rostovtseva T., et al, 2008

22. Interaction with hexokinase • Aerobic glycolysis (Warburg effect) is a typical feature ofcancer cell metabolism, which is characterized by high aerobicglycolysis, suppression of mitochondrial respiration and highexpression of hexokinase • Hexokinase, the first enzymein the glycolytic pathway, binds to the mitochondrial outermembrane via a specific association with VDAC1. • Hexokinase-I acting through its N-terminal mitochondrialbinding domain blocks conductance of rat livermitochondrial VDAC reconstituted into lipid bilayers Shown on the reconstructed protein system, but not in the intact mitochondria! The Aim of this work is to investigate functional state of VDAC in intact mitochondria and especially its interaction with hexokinase using Peak Intensity Analysis approach.

23. ATP - hydrophilic molecule TMREpositively TMRM charged Rh123arehydrophobic JC-1 fluorescent Mitotracker…..dyes ATP-Bodipy fluorescent marker + + _ _ VDAC Hydrophilic fluorescent probe is used for mitochondrial study

24. Peak Intensity Analysis (PIA) 10 mM ATP 10 mM ATP 10 mM ATP 100 mM ATP 100 mM ATP 1 mM ATP Perevoshchikova et al, BBA, 2008

25. Interaction ATP-BP with lipids 25 nM ATP-Bodipy, liposomes: 0.1 mg/ml brain phosphatidylserine (PS), 0.1 mg/ml azolectin (Azo), 0.1 mg/ml Egg L-a-Phosphatidylcholine (PC). Buffer: 100 mM KCl, 10 mM MES, 10 mM Tris, pH=7.0

26. ATP-Bodipy is mostly accumulated in mitochondria through VDAC channel NADH, König‘s Polyanion + + + Closed state of VDAC Open state of VDAC König‘s polyanion NAD+, NADH

27. Isolated hexokinase II binding to VDAC channel blocks ATP-Bodipy flux to mitochondria Perevoshchikova et al, FEBS Letters, 2010

28. 15-residue peptideMIASHLLAYFFTELN-amidecorrespondingto the N-terminal domain of hexokinase does not reduce the accumulation of ATP-Bodipy to mitochondria Perevoshchikova et al, FEBS Letters, 2010

29. Conclusions • FCS is very sensitive technique due to extremely low concentration of fluorescent molecules needed for measurement and single photon detecting system