UNIVERSITA’ DEGLI STUDI
DI BARI
FACOLTA’ DI SCIENZE MATEMATICHE FISICHE E
NATURALI
Studio dei segnali di trasduzione in
cellule in vivo: applicazione della
microscopia FRET
The Fluorescence Process
Stage 1: Excitation
Stage 2: Excited-State
Lifetime
Stage 3: Fluorescence
Emission
The process responsible for the fluorescence properties of fluorescent
probes and other fluorophores is illustrated by the simple electronic-state
diagram called a Jablonski diagram.
Excitation and Fluorescence Emission Spectra
Shift di Stokes
Fluorescence
excitation spectrum
Fluorescence emission
spectrum
The entire fluorescence process is cyclical.
Different types of light and their associated wavelengths
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Fluorescence
excitation spectrum
Fluorescence emission
spectrum
Probes for Proteins
Probe
FITC
PE
APC
PerCP
Cascade Blue
Coumerin-phalloidin
Texas Red
Tetramethylrhodamine-amines
CY3
CY5
Excitation
488
488
630
488
360
350
610
550
540
640
Emission
525
575
650
680
450
450
630
575
575
670
Probes for Nucleic Acids
•
Hoechst 33342 (AT rich) (uv)
346
460
•
DAPI (uv)
359
461
•
POPO-1
434
456
•
YOYO-1
491
509
•
Acridine Orange (RNA)
460
650
•
Acridine Orange (DNA)
502
536
•
Thiazole Orange (vis)
509
525
•
TOTO-1
514
533
•
Ethidium Bromide
526
604
•
PI (uv/vis)
536
620
•
7-Aminoactinomycin D (7AAD)
555
655
GFP is from the chemiluminescent jellyfish
Aequorea victoria
Green Fluorescent Protein
AEQUOREA VICTORIA
GFP
• Discovered as companion protein to Aequorin (blue fluorescent protein)
• The components required for bioluminescence include a photoprotein,
aequorin, that emits blue-green light, and an accessory green fluorescent
protein (GFP), wich accepts energy from aequorin and re-emits it as green
light
Fluorescent protein spectral variants
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emission
1.0
eGFP
eCFP
eYFP
DsRed
0.5
0.0
400
450
500
550
600
nm
1.0
excitation
650
0.5
0.0
400
450
500
550
600
650
nm
Fluorescent proteins as:
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• probes to monitor proteins colocalization
• molecular markers to track and quantify individual
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or multiple protein species (in vitro / in vivo)
• photo-modulatable proteins to highlight and follow the fate of specific protein
populations
FRET
• Permette l’osservazione di interazioni molecola-molecola nel range di nm
• Trasferimento di energia non radiattivo da un fluorocromo (donatore) ad un altro
(accettore) quando essi si trovano vicini tra loro (quenching)
• Definito anche Förster Resonance Energy Transfer
• Osservato per la prima volta proprio in Aequorea victoria tra aequorina e GFP
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THE PRINCIPLE OF FRET
480 nm
480 nm
430 nm
D
Max 100 Å
D
2)
The probe A absorbs at
480nm and emits at 545nm
3)
When these protein are
brought in close proximity,
energy transfer can occur (red
row).
A
The probe D is excited by
absorbing light at 430nm and
transfers the energy to probe A.
A
545 nm
EM EX.
.
The probe D absorbes light at
430nm and emits light at
480nm
545 nm
EX. EM
.
EX. EM
. 430 nm
1)
Now the probe A is excited and
falls back to its ground state,
thereby emitting light at 545nm.
When D and A are in close proximity, the emission at 480 nm is decreased and the
emission at 545nm is increased.
FRET
• Il FRET intramolecolare avviene
quando sia donatore che accettore
sono fusi con la stessa molecola
che subisce una transizione
(cambiamento di conformazione)
L’efficienza
di
FRET
dipende
dall’orientamento relativo e dalla
distanza tra donatore e accettore
• Il FRET intermolecolare avviene tra
una molecola (Protein A) fusa con il
donatore
e
un’altra
molecola
(Protein B) fusa con l’accettore.
Quando
le
due
proteine
interagiscono
si
osserva
il
fenomeno di FRET
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GFP Mutants
…..what are the best
FRET partners?
Primary conditions for FRET microscopy
•Donor and Acceptor molecules must
be in close proximity (tipically 10-100
A)
•Donor and Acceptor transition dipole
orientations must be approximately
parallel
•Absorption spectrum of the acceptor
must overlap the fluorescence
emission spectrum of the donor (see
figure on the left)
Known FRET pairs are CFP/YFP, BFP/GFP, GFP/Rhodamine, FITC/ Cy3
Fluorescence resonance energy transfer
CFP
YFP
Exc: 430 nm
Em: 480 nm
Exc:475 nm
Em: 545 nm
FRET
• Alle cellule vengono fatte
esprimere le proteine di interesse
attraverso una singola trasfezione
(intramolecolare)
o
una
cotrasfezione (intermolecolare).
• Per osservare il fenomeno di
FRET il campione viene eccitato
nella λ di eccitazione del donatore
• Si registra il segnale emesso dal
campione sia nella λ di emissione
del donatore che dell’accettore.
Se vi sono le condizioni ideali si
osserva una diminuzione del
segnale relativo al donatore ed un
aumento
di
quello
relativo
all’accettore.
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FRET MICROSCOPE
Beamsplitter (2 Detectors)
sample
objective
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Excitation
CFP (430 nm)
Dicroic (455 nm)
Detector
CFP
Emission CFP
(480nm)
Dicroic (505 nm)
Emission YFP
(545)
Detector
YFP (FRET)
The ratio of Donor to Acceptor emission has been
used as an index of the extent of FRET
FRET
FRET
430 nm
nm
430
FRET APPLICATIONS:
 Variations in membrane potential
 Protease activity
 Variations in intracellular Ca2+ and cAMP
levels
 Conformational protein changes
 Protein-protein interactions
Cyclic AMP signalling
From: G.M. Fimia and P. Sassone-Corsi, Journal of Cell Science 2001
Invasive signals from the microenvironment
act as inducer of tumor progression
Cell migration in tumors
•Collective motility
(solid cell strands, sheets, files
and clusters)
•Amoeboid motility
Leading-edge
rich in small
pseudopodia (shape-driven migration
Trailing
uropodia
Ellipsoid cell
body
path
finding)
•Mesenchimal motility
Extrinsic factors from the
elongated and
adhesive phenotipe
Surface protease
Leading-edge
Direction of movement pseudopodia
path generation
microenvironment can promote tumor motility!
MCF10-A
Normal, human
breast cell line
MDA-MB-435
Human breast
metastatic cell line
Invasion specific Signal Transduction
cascade
Increased invasive
capacity
(+)
cAMP
NHE1
p38
PKA
P
RhoA
ROCK
Serum deprivation redistributes total RhoA, phospho
RhoA, NHE1 in to MDA-MB-435 pseudopodial
compartment
Are the mobilization of cAMP and activation of PKA
localized to the leading edge pseusopodia of cancer cells?
NHE1
Local pool
of cAMP?
p38
Local pool
of PKA?
P
ROCK
RhoA
How this cAMP-mediated signalling is modulated
by invasion promoting stimuli?
THE cAMP SENSOR IS A CHIMAERIC PROTEIN KINASE A
535nm
FRET
YFP
CFP
535nm
CAT
CAT
CAT
YFP
R
FRET
R
CFP
480nm
430nm
430nm
480nm
GPCR
g
a
b
AC
ATP
cAMP
Inactive PKA
CAT
CAT
R
CAT
R
GPCR
g
a
AC
b
CAT
Active PKA
CAT
R
R
How is it possible to measure cAMP with FRET?
FRET
YFP
CFP
CAT
CAT
CAT
YFP
R
Low cAMP 430nm
R
CFP
430nm
FRET
YFP
YFP
CAT
CAT
CA
CA
YFP
T
T
CFP
CFP
cAMP
R
R
430nm
R
Reg.
CA
YFP
T CF
R
P
CFP
430nm
Zaccolo M. et al., Science ,2002.
cAMP sensors based on PKA
pBI-cRII-ycat
YFP
RII
CAT
CFP
c
pBI-RII-myrpalm
YFP
CAT
c
mp
RII
CFP
EPAC H30
Ponsioen et al.
EMBO Rep. 2004 December; 5(12): 1176–1180
myrpalm
HBE
cytosolic
HBE
A-Kinase activity reporter (AKAR)
Ni Q, Titov DV, Zhang J.
Methods. 2006 Nov;40(3):279-86.
cytosolic
myrpalm
1,15
1
***
1,15
EmCFP/EmYFP
EmCFP/EmYFP
Detection of local cAMP activity using FRET in
both MCF10-A and MDA-MB-435 cells
*
**
**
1
0,9
0,9
mb
psu
ND
mb
psu
mb
psu
ND
D
MCF10 A
mb
psu
D
MDA-MB-435
High cAMP
concentration
RII-CFP EmCFP/EmYFP
Low cAMP
concentration
High cAMP
concentration
RII-CFP EmCFP/EmYFP
Low cAMP
concentration