Fluorescence in the Study of Fluorescence in the Study of Cancer: From the Molecular to the Cancer: From the Molecular to the

MacroscopicMacroscopic

Presented to ACCA on November 3, 2009Seminar on Biological Fluorescence

ByJuanita C. Sharpe

Associate ProfessorChicago State University

Why Do We Get Cancer?Why Do We Get Cancer?

• Mutations acquired through aging

– Multiple mutations lead to cancer

• Old DNA is genetically unstable

– Inaccurate replication of the genome• 10-6 – 10-7 mutations/gene/cell division – 1016

cell divisions probability of 109 mutations/gene

– Inefficient DNA repair– Increases in chromosome breakage and

rearrangements

Cancerous Changes Can Result From Cancerous Changes Can Result From Environmental DamageEnvironmental Damage

h

h

Cancerous Changes Can Result From Cancerous Changes Can Result From Environmental DamageEnvironmental Damage

Damage to a Dividing Cell is the First Damage to a Dividing Cell is the First Step On the Road to Cancer Step On the Road to Cancer

As the damaged cell continues to divide it’s effects spread

Damage to a Dividing Cell is the First Damage to a Dividing Cell is the First Step On the Road to Cancer Step On the Road to Cancer

Each new division may result in the acquisition of new mutations.

Why Don’t We All Get Cancer During Why Don’t We All Get Cancer During Our Lifetimes?Our Lifetimes?

• DNA damage and ageing leads to cancerous changes

• Environmental exposure lead to cancerous changes

Programmed Cell Death!

Morphological and Biochemical Features of Programmed Cell Death Morphological and Biochemical Features of Programmed Cell Death (Apoptosis)(Apoptosis)

Degradation Phase

Phosphatidylserine

Blebs

ProteolysisEndonucleases

Nucleus

Initiation Phase

Death Signals: • Oxidative Stress• Glutamate• Decreased Growth Factors• Genetic Mutation

Oxygen Radicals/Ca2+

stimuli

Bad/Bax

Mitochondrion

Effector Phase

Cytochrome CAPAF-1Caspase-9

Caspase-3

DeathSubstrates

Nucleus

h

Cancerous Changes Can Result From Cancerous Changes Can Result From Environmental DamageEnvironmental Damage

The Bcl-2 familyThe Bcl-2 family

BH4 BH3 BH1 BH2 TM

Bcl-2

Bcl-xL

Bcl-w

Bax subfamily

Bak

BH3-only subfamily

Bid

Bad

Bik

Blk

Hrk

Bim

Bcl-2 subfamily

Pro-survival

Pro-apoptotic

*

Bax *

*

Bcl-2 Family Members Act at the Mitochondrial MembraneBcl-2 Family Members Act at the Mitochondrial Membrane

SurvivalDeath

Bax

BaxBax

cardiolipin

cyt. c

AIF

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

BaxBcl-Xl

Contact Sites

Fulcrum

BaxBcl-2

Bcl-Xl

BaxBid

Caspase 8

Mitochondria

t-bid

Bax

Bcl-2

The Mitochondrial Pathway of Apoptosis The Mitochondrial Pathway of Apoptosis InitiationInitiation

Caspase 3

Caspase 3*

CADICAD

CADICADcleaved

Substrates:PARP, Lamin,Fodrine, FAK,PAK-2, PKC…

IAPs

AIF

Endo G

Caspase 8*

Mitochondria

Bid

Bax/Bak Bcl-2Bcl-XL

Bax

Drugs, U.V,Deprivation ...

Smac + HtrA2Cytochrome c

Apaf-1 Caspase 9

"Apoptosome"Caspase 9*

D. Arnoult

Apoptosis at the MitochondriaApoptosis at the Mitochondria

Caspase 3

Caspase 3*

Mitochondria

Bcl-2Bcl-XL

Bax

Cytochrome c

Apaf-1 Caspase 9

"Apoptosome"Caspase 9*

Chews up the inside of the cellThe cell dies

Bid, In Cooperation with Bax Disrupts Mitochondrial MembranesBid, In Cooperation with Bax Disrupts Mitochondrial Membranes

Death

Bax

BaxBax

cardiolipin

cyt. c

AIF

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

Contact Sites

Fulcrum

BidCaspase 8

Mitochondria

t-bid

Bax

Bcl-2

Cancer, Bax and Programmed Cell Death as Cancer, Bax and Programmed Cell Death as a Model Systema Model System

Bax

cyt. cAIF

Bax

Contact Sites

cardiolipin

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

AIF

cyt. c

Mitochondria

Bax

VDACBax

BaxBax

•Hypothesis: The interaction between Bax and cardiolipin is required to induce apoptosis

•Do these two molecules structurally and functionally interact?•Where does cardiolipin interact with Bax?•How does this interaction affect the structure of Bax?•Are the structural changes functionally significant?•Is this interaction involved in regulating apoptosis?

How Do We Study The Relationship How Do We Study The Relationship Between Structure and FunctionBetween Structure and Function

• Combination of techniques addresses structure and function– Fluorescence techniques

• Structural and functional changes• Isolate variations in function

– Biochemical/cellular techniques• Functional changes in the mitochondria and/or cell• Mutational analysis to determine changes in

function of Bax

Principles of FluorescencePrinciples of Fluorescence

• If the photon emission occurs between states of the same spin state (e.g. S1 ---> S0) this is termed fluorescence.

• The lifetimes of fluorescent states are very short (1 x 10-5 to 10-8 seconds)

Jablonski Diagram

Mechanisms to Study Protein StructureMechanisms to Study Protein Structure• Tryptophan is an inherently fluorescent amino acid• Tryptophan fluorescence is sensitive to the surrounding

environment• We use tryptophan fluorescence to perform structure

function studies

0

50000

100000

150000

200000

250000

320 340 360 380 400 420

Wavelength

Inte

nsi

ty Normal fluorescence

Blue Shift

Red Shift

350

340

360

Bax

Tryptophan fluorescence is an average of water exposed and buried residues

= 350

More tryptophans are buried away from the water

= 340

Bax Bax

Tryptophans have moved away from the interior of the protein to become water exposed. = 360

Bax in solution Bax in the membrane Unfolded Bax

Idealized “Classical” Bax BehaviorIdealized “Classical” Bax Behavior

Unique Conformational Changes As Unique Conformational Changes As Monitored In Model MembranesMonitored In Model Membranes

lipid 7

lipid 5

Bax 7

PositiveInsertion control

320 340 360 380 400

349 nm

340

346

Negative control

320 340 360 380 400

Wavelength

-1.00E+06

1.00E+063.00E+06

5.00E+067.00E+06

9.00E+061.10E+07

1.30E+071.50E+07

1.70E+07

Inte

nsity

348

Wavelength

349 nm

Experimental

320 340 360 380 400

Wavelength

344

344

-1.00E+06

1.00E+063.00E+06

5.00E+067.00E+06

9.00E+061.10E+07

1.30E+071.50E+07

1.70E+07

Inte

nsity

349 nm

Classical Insertion Behavior

Bax Bax

Novel conformation not explained by classical insertion behavior.

Positive Insertion Control

ExperimentalConditions

Experimental ObservationsExperimental Observations

Principles of Fluorescence QuenchingPrinciples of Fluorescence Quenching

• Quenching can occur through a variety of methods– Collisional quenching – – Static quenching – – Anisotropic quenching – – Resonance Energy Transfer –

Static QuenchingStatic Quenching

• when a molecule contacts another molecule that has the ability to fluoresce and prevents the electron from getting to the excited state– May be due to the formation of non fluorescent

complexes between fluorophores and quenchers– Occurs in the ground state– Does not rely on diffusion or molecular collisions

How to Obtain Detailed Information How to Obtain Detailed Information About Bax Conformation In Membranes?About Bax Conformation In Membranes?

Shallow(Tempo)

Medium(5-doxyl)

Deep(12-doxyl)

Quenchers in the Membrane

Protein

Pro

tein

Protein

• SUVs containing one of each quencher are made• One Sample does not contain quencher• 4 Samples total for each experiment

Quenching is determinedBy the equation 1- (Fq/Fo)

Static Quenching Detects Differences in Static Quenching Detects Differences in Protein Insertion BehaviorProtein Insertion Behavior

pH 7.4 pH 5.2 pH 4.0

Positivecontrol

Experi-mental

Positivecontrol

Experi-mental

Positivecontrol

Shallow 21% 2.7% 9.3% 0% 13%

Medium 19% .6% 22% 0% 29%

Deep 16% 6.8% 34% 0% 43%

Explanation of Differences in Control Explanation of Differences in Control and Experimental Membranesand Experimental Membranes

PGPC PGPC PC PG

BaxBa

x

BaxpH 7

PGPC PGPC PC PGBax

Bax

Bax

PGPC PGPC PC PG

Bax

Bax

Bax

pH 5

pH 4

Bax

PCClPC PCClClPC

Bax

Cl Cl ClBaxPC PCPC

Insertion Control Experimental

Fluorescence Can be Use to Study FunctionFluorescence Can be Use to Study Function

• Caged fluorescent dextrans have been use to Caged fluorescent dextrans have been use to measure pore size or membrane disruption measure pore size or membrane disruption

• Quenching of dextrans of various sizes can indicate Quenching of dextrans of various sizes can indicate the size of a pore formed by a protein in the the size of a pore formed by a protein in the membranemembrane

• Does Cardiolipin aid in pore formation/vesicle Does Cardiolipin aid in pore formation/vesicle rupture?rupture?

Dextran Release Measures Channel Dextran Release Measures Channel FormationFormation

BAX

BAX

BAX

Decrease pH

BAX

BAX

Y

Y

Y

Y

Y

Y

Y

Y

Fluorescent Non-fluorescent

Y

Cardiolipin Enhances rBax Mediated 3 kD Cardiolipin Enhances rBax Mediated 3 kD Dextran ReleaseDextran Release

pH

0

20

40

60

80

100

7.70 7.00 6.40 5.00

% M

axim

al Q

uen

chin

g

Yellow = 30% Cardiolipin LUVPink = 30% DOPG LUV

Model for the Mechanism of Bax Model for the Mechanism of Bax Interaction with the MitochondriaInteraction with the Mitochondria

6A7

Bax

Bax

Bax Bax

Mitochondria

6A7

Bax

IMS Protein Release

Activation of Cell Death

?

What Regions of Bax are Involved in the What Regions of Bax are Involved in the Cardiolipin InteractionCardiolipin Interaction

BH3

BH1 BH2 CTBax

•The C-terminal (CT) helix of Bax is important for membrane insertion, does it also contribute to cardiolipin binding?

•Created a mutant missing the c-terminal helix

BH3

BH1 BH2Bax C

Truncated Bax Responds to Cardiolipin in Truncated Bax Responds to Cardiolipin in the Same Manner as Full Length Baxthe Same Manner as Full Length Bax

Losing the hydrophobic tail does not inhibit cardiolipin binding at neutral pH

Truncated Bax Responds to Cardiolipin in Truncated Bax Responds to Cardiolipin in the Same Manner as Full Length Baxthe Same Manner as Full Length Bax

Nor does the loss of the hydrophobic tail inhibit cardiolipin binding at low pH

Other Uses for Fluorescence in Studying CancerOther Uses for Fluorescence in Studying Cancer

• Fluorescence is not only used to study protein conformational changes and interactions with membranes

• Fluorescence is increasingly used to:– image cells - confocal microscopy – Study intermolecular interactions– Genetic interactions– Track and image metastatic cancers in whole organisms

Resonance Energy TransferResonance Energy Transfer

• Energy transfer occurs between a donor fluorescent molecule and an acceptor molecule (can be fluorescent or non-fluorescent)

• The two molecules have overlapping spectra• The donor light emission must overlap the

acceptor absorption spectra• The acceptor can use that energy to fluoresce

at a different wavelength than the donor

• The energy of the fluorescent donor can be used to excite the acceptor molecule

• That energy can no longer be used by the fluorescent molecule for fluorescence

• Instead the energy is absorbed by the other molecule

Resonance Energy TransferResonance Energy Transfer

• In FRET the fluorescence of the acceptor is often used as a measure of energy transfer

Resonance Energy TransferResonance Energy Transfer

The Principles of FRETThe Principles of FRET

• Molecules must be at least 10 nm apart.• The dipole of the fluorophores must not be at right

angles to each other• The concentrations of donor and acceptor

fluorophores must be closely controlled. – The statistically highest probability of achieving

fluorescence resonance energy transfer occurs when a number of acceptor molecules surround a single donor molecule.

The Principles of FRETThe Principles of FRET• Photo bleaching must be eliminated because the

artifact can alter the donor-to-acceptor molecular ratio, and therefore, the measured value of the resonance energy transfer process.

• There should be minimal direct excitation of the acceptor in the wavelength region utilized to excite the donor. A common source of error in steady state FRET microscopy measurements is the detection of donor emission with acceptor filter sets.

• The donor absorption and emission spectra should have a minimal overlap in order to reduce the possibility of donor-to-donor self-transfer.

The Principles of FRETThe Principles of FRET

• The donor molecule must be fluorescent and exhibit sufficiently long lifetime in order for resonance energy transfer to occur.

• The donor should exhibit low polarization anisotropy to minimize uncertainties in the value of the orientation (k-squared) factor. This requirement is satisfied by donors whose emission results from several overlapping excitation transitions.

The Principles of FRETThe Principles of FRET

• Because fluorescence resonance energy transfer requires the donor and acceptor molecules to have the appropriate dipole alignment and be positioned within 10 nanometers of each other, the tertiary structure of the reagents to which the molecules are attached must be considered. – For example, when donor-acceptor molecules can be

attached to different structural locations (such as the carboxy or amino terminus) on a protein, it is possible that FRET will not be observed even though the proteins do interact, because the donor and acceptor molecules are located on opposite ends of the interacting molecules.

Types of FRETTypes of FRET

• Types of FRET– Sensitized emission

• Do full scans of the excitation and emission of the donor and acceptor fluorophores and pick excitation wavelengths and emission wavelengths that favor donor excitation and acceptor emission

– Acceptor photo bleaching FRET• A way to determine energy transfer efficiency and quantify the

amount of background “noise” from the samples

Emission Spectra of Cy2 and Cy3Emission Spectra of Cy2 and Cy3Notice the overlap between the emission spectra of Cy2 and Cy3

The further you move away from the excitation peak the more weak your donor fluorescence will be and the lower your energy transfer efficiency will be

Measuring the fluorescence of Cy2 at the peak of emission will allow you to pick up fluorescence from Cy3

FRETFRET• The excitation and emission spectra for Cy2 are close • The excitation and emission spectra for Cy3 are close

(small stokes shift)– Stokes shift is the difference in energy of excitation and

emission of the fluorophores• Ideally like to choose a fluorophores whose excitation

spectra is far from its emission spectrum• Otherwise there is lots of “bleed through” between the

signals– Get background fluorescence from the donor near the acceptor

wavelength– Get background excitation of the acceptor from the donor

excitation wavelength

Acceptor Photobleaching FRETAcceptor Photobleaching FRET

• Used to calculate efficiency of energy transfer• Can calculate how close the molecules are to each

other• Confirms that energy transfer occurred between

donor and acceptor– If the donor fluorescence increases when the acceptor

is photobleached then the decrease in fluorescence in the original sample was due to energy transfer

• Helpful in fluorescent microscopy when the change in fluorescence is small

Acceptor Photobleaching FRET demonstrates Acceptor Photobleaching FRET demonstrates the Phosphorylation state of the EGF Receptorthe Phosphorylation state of the EGF Receptor

• EGF receptor phosphorylation was studied using Cy3-labeled EGF receptor and a Cy5 labeled antibody against phosphotyrosine

• Green = Cy3 labeled EGRF• Red = Cy5 labeled anti

phosphotyrosine

• Interaction was demonstrated between these two molecules by an increase in fluorescence of Cy3 after photobleaching

Keese M. et. al. JBC 2005; v. 280 (30)

Uses of FRETUses of FRET

• Study conformational changes by measuring energy transfer efficiencies thereby obtaining changes in distances

• Measuring interacting proteins and can provide quantitative measurements of the distances of interactions

• Measuring the oligomeric state of proteins

Fluorescence Lifetime Imaging Fluorescence Lifetime Imaging Microscopy (FLIM)Microscopy (FLIM)

• Fluorescence lifetime – the amount of time a molecule spends in the excited state

• Fluorescent lifetime is affected by – Solvent polarity– pH of the solution– Ion concentration– Energy transfer

FLIMFLIMHigher intensity readings indicate the lack of energy transfer because the fluorescent molecule stays in the excited state longer

Lower intensity readings indicate energy transfer because the molecule is in the excited state for a shorter amount of time

FLIM Demonstrates the Interaction between FLIM Demonstrates the Interaction between EGFR and Anti-phosphotyrosine MoleculesEGFR and Anti-phosphotyrosine Molecules

• Blue color indicates a shorter lifetime – Shorter lifetime molecules have higher energy and shorter

wavelengths• Green color indicates a longer lifetime• After photobleaching the fluorescence lifetime of Cy3

increasesKeese M. et. al. JBC 2005; v. 280 (30)

Collisional QuenchingCollisional Quenching• when one molecule collides with a fluorescent

molecule and upon contact removes the electron from the excited state decreasing its ability to fluoresce

Collisional quenching is most likely due to electron transfer in the excited state

Acrylamide Collisional Quenching Indicates Acrylamide Collisional Quenching Indicates Molecular Exposure to the Membrane SurfaceMolecular Exposure to the Membrane Surface

PC PC

PCPC

PGPG CLCL

PGPG CLCL

= less fluorescence remaining/more quenching

= more fluorescence remaining/ less quenching

= greater fluorescence remaining/ least quenching

AA

A

A

A

A

A

A

AA

A

A B C

Expected Results with Acrylamide Expected Results with Acrylamide QuenchingQuenching

% Q

uen

chin

g

[Acrylamide] [Acrylamide] [Acrylamide]

A B C

Acrylamide Quenching of Resveratrol Acrylamide Quenching of Resveratrol FluorescenceFluorescence

• Resveratrol has potential as an anti-cancer compound.

• When incorporated into artificial vesicles, resveratrol fluorescence is resistant to fluorescent quenching by acrylamide (open circles)

• Resveratrol inhibits Protein Kinase C, a protein involved in cell cycle regulation

Garcia-Garcia et. al. 1999 Archives of Biochemistry and Biophysics vol. 372 pp. 382-388

Fluorescence Correlation Fluorescence Correlation SpectroscopySpectroscopy

• Image contrast is based on the time the fluorescent molecule is in the excited state.

• Molecules experiencing longer lifetimes show lower contrast (more easily quenched) to the background than molecules with longer lifetimes (less easily quenched) stronger signal

• Different lifetimes equal different environments of the fluorophore.

• Fluorescent lifetimes are measured under 10-9 seconds

Fluorescence Correlation Fluorescence Correlation SpectroscopySpectroscopy

• Used to calculate parameters such as: – diffusion coefficients – hydrodynamic radii – average concentrations – kinetic chemical reaction rates – Conformational changes in a time specific manner

• Uses fluorescence lifetime of a single molecule to obtain information about the environment of the fluorophore

Fluorescence Correlation Fluorescence Correlation Spectroscopy Spectroscopy

• Uses small volumes to measure single molecule fluorescence

• Based on calculating the concentration of fluorophore in the total sample and observing a small enough volume to get one fluorophore per sample

• Mobility of the sample can be measured and can indicate parameters such as ligand binding

• The difference between ligand and receptor has to be different by a factor of 4 because of diffusion rate differences (1.6)

• Uses a single fluorophore

FCS determines Concentration and FCS determines Concentration and MobilityMobility

• Confocal volume is the volume of the sample measured

• G is the correlation time

• is the time the fluorophore is in the measured state

• Molecular interactions cause the molecule to stay in each phase of the excited state for a shorter amount of time

Fluorescence Cross Correlation Fluorescence Cross Correlation SpectroscopySpectroscopy

• Uses two fluorescent probes to determine the interaction between two molecules

• Less sensitive to molecule size• Both FCS and FCCS are measured in live cells• Time-correlated single photon counting

– This method is used to analyze the relaxation of molecules from an excited state to a lower energy state.

– Since various molecules in a sample will emit photons at different times following their simultaneous excitation, the decay must be thought of as having a certain rate rather than occurring at a specific time after excitation.

– By observing how long individual molecules take to emit their photons, and then combining all these data points, an intensity vs. time graph can be generated that displays the exponential decay curve typical to these processes. Individual excitation-relaxation events are recorded and then averaged to generate the curve.

FCCS and Molecular InteractionsFCCS and Molecular Interactions• Measure diffusion rate of two molecules in a small volume• Measure the fluctuations in intensity and how they correlate

with each other to determine binding• If the molecules interact the fluctuations occur near the same

time and the fluctuations of each correlate with each other

FCCS and Molecular InteractionsFCCS and Molecular Interactions• If the two molecules do not interact this means that their

fluoresce is in the area of measurement at different times. These fluorescent fluctuations do not correlate with each other and their cross correlation value is low

Many Ways to Use Fluorescence to Study Many Ways to Use Fluorescence to Study CancerCancer

• Total internal reflectance fluorescent microscopy– Enables the study of single molecules within a narrow

band using evanescent waves

• Fluorescence Polarization– Enables the study of the diffusion and/or rotation of

molecules

• Fluorescence Activated Cell Sorting (FACS) – uses fluorescence to tag and isolate cells containing specific fluorescent signals

Near Infrared Fluorophores are Being Used to Near Infrared Fluorophores are Being Used to Image Cancers in VivoImage Cancers in Vivo

Ogawa M. et. al. Cancer Research 2009; 69 (4)