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

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

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

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

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

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

Calculating the Autocorrelation Function time Photon Counts t  t +  Average Fluorescence t1t1 t2t2 t3t3 t4t4 t5t5

The analytical form of the autocorrelation function 3 D diffusion, one-photon excitation r 0 =0,42  m z 0 =2,1  m V conf =2,2 fl  d – diffusion time D – diffusion coefficient average brightness per molecule - average intensity of fluorescence -

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

The Effects of Particle Size on the diffusion time 300 um 2 /s 90 um 2 /s 71 um 2 /s Diffusion Constants Fast Diffusion Slow Diffusion Stokes-Einstein Equation: and Monomer --> Dimer Only a change in D (hence  d ) by a factor of 2 1/3, or 1.26 Fast diffusion Slow diffusion

The Autocorrelation functions of mitochondria, liposomes and fluorescent dyes Mitochondria

Trace of fluorescence and Photon Counting Histogram (PCH) Stirring condition 1. Non-energized state 2. Energized state 3. Deenergized state Tetramethylrhodamine, ethyl ester, TMRE 1.TMRE 2.TMRE 0,03 μМ, mitochondria, rotenone 3.Addition of succinate 4.Addition of DNP + succinate + DNP Δφ≈0 Δφ≈-200 мВ Δφ≈0 PCH Trace of fluorescence 1 MHZ 50 ms Probability/bin Fluorescence intensity, kHz

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

Trace of fluorescence of identical fluorescent spheres (1  m in diameter)

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

Theoretical background z0z0 r0r0 z x Quasi-cylindrical profile of the observation volume can be described by a Gaussian distribution of the detected intensity [Rigler R. et al. 1993]: B 0 – 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/r 0, y= z/z 0, and R=(x 2 +y 2 ) 1/2. Then, the maximal intensity will be: The portion of events having the amplitude more than F 0 (i.e., P(F>F 0 )) is R 2 /R 0 2, where R 0 is a radius of a circle in the x,y space, where all events take place. By expressing R through F we obtain: forandfor Experimentally not probability P(F>F 0 ), but number of events with fluorescence intensity higher than the definite level N(F>F 0 ) was measured and N(F>F 0 )~P(F>F 0 ). P 0 – factor which is proportional to number of particle in suspension

Fluorescent beads, d=1  m F initial F0F0 amplitude N(F>F 0 ) F 0, kHz Gaussian distribution Lorentzial distribution Derivative of P(F>F 0 ) gives the number of events in some interval around F=F 0 and easily might be obtained by differentiation of equation : (Gaussian distribution) In some cases of one-photon excitation profile of the observation volume can be described by a Lorentzial distribution [Hess S., Webb W., 2002]: Then function P(F>F 0 ) and its derivative are: forandfor (Lorentzial distribution) (Gaussian- Lorentzial distribution) Win EDR

Peak Intensity Analysis (PIA) F 0, kHz N(F>F 0 ) TMRE, nM Р(F>F 0 ) - probability to detect particle with fluorescence value upper than F 0 Р 0 - factor proportional to particle concentration in suspension А∙В 0 – brightness of particle in center of confocal volume Suspension of latex beads (d=0,8 μm) doped with different concentration of TMRE N(F>F 0 ) F 0, кГц Suspension of fluorescent particles 1.d=1 μм, А∙В 0 = 11700 kHz 2.d=0,5 μм, А∙В 0 =1600 kHz 3.d= 0,1 μм, А∙В 0 =1080 kHz From autocorrelation function, N does not depend on stirring 1.d=1 μм, =11000 kHz 2.d=0,5 μм, =5600 kHz 3.d= 0,1 μм, =2100 kHz N(F>F 0 )~P(F>F 0 )

One particle detection condition Fluorescent beads d=0,5 μм Energized mitochondria doped with TMRE Protein concentration, mg/mlNumber of particles, particles/ml

PIA application to suspension of mitochondria doped with TMRE 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 F 0, kHz 1 – mitochondria, rotenone А∙В 0 =1880 kHz, Р 0 =400 2 – addition of succinate А∙В 0 =6600 kHz, Р 0 =540 3 – addition of DNP А∙В 0 =2760 kHz, Р 0 =314 N(F>F 0 ) 4000 kHz ms N(F>F 0 ) F 0, kHz PCRh-PE liposomes АВ о = 750 кHz S of lipid molecule= 0.7 nm 2 Diameter of liposome = 80 nm (Zsizer Nano) N rhodamine = 574 in one liposome 0,0044 mg/ml mitochondrial protein

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

Voltage Dependent Anion Cannel (VDAC) Ujwal R. et al, 2008 Rostovtseva T., 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)

Interaction with hexokinase 1.Aerobic glycolysis (Warburg effect) is a typical feature of cancer cell metabolism, which is characterized by high aerobic glycolysis, suppression of mitochondrial respiration and high expression of hexokinase 2.Hexokinase, the first enzyme in the glycolytic pathway, binds to the mitochondrial outer membrane via a specific association with VDAC1. 3.Hexokinase-I acting through its N-terminal mitochondrial binding domain blocks conductance of rat liver mitochondrial 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.

Hydrophilic fluorescent probe is used for mitochondrial study ATP-Bodipy ATP - hydrophilic molecule _ + TMREpositively TMRM charged Rh123 are hydrophobic JC-1 fluorescent Mitotracker….. dyes _ + fluorescent marker VDAC

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

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

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

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

15-residue peptide MIASHLLAYFFTELN-amide corresponding to the N-terminal domain of hexokinase does not reduce the accumulation of ATP-Bodipy to mitochondria Perevoshchikova et al, FEBS Letters, 2010

Conclusions 1.FCS is very sensitive technique due to extremely low concentration of fluorescent molecules needed for measurement and single photon detecting system

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