UNIVERSITA’ DEGLI STUDI DI BARI

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1 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

2 The process responsible for the fluorescence properties of fluorescent
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.

3 Excitation and Fluorescence Emission Spectra
Shift di Stokes Fluorescence excitation spectrum Fluorescence emission spectrum The entire fluorescence process is cyclical.

4 Fluorescence emission spectrum
Different types of light and their associated wavelengths Fluorescence emission spectrum Fluorescence excitation spectrum

5 Probes for Proteins Probe Excitation Emission FITC 488 525 PE 488 575
APC PerCP Cascade Blue Coumerin-phalloidin Texas Red Tetramethylrhodamine-amines CY CY

6 Probes for Nucleic Acids
Hoechst (AT rich) (uv) DAPI (uv) POPO YOYO Acridine Orange (RNA) Acridine Orange (DNA) Thiazole Orange (vis) TOTO Ethidium Bromide PI (uv/vis) 7-Aminoactinomycin D (7AAD)

7 GFP is from the chemiluminescent jellyfish
Aequorea victoria

8 Green Fluorescent Protein
GFP AEQUOREA VICTORIA 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

9 Fluorescent protein spectral variants
0.0 1.0 0.5 450 500 550 600 650 400 eGFP emission excitation nm eCFP DsRed eYFP

10 Fluorescent proteins as:
probes to monitor proteins colocalization molecular markers to track and quantify individual or multiple protein species (in vitro / in vivo) photo-modulatable proteins to highlight and follow the fate of specific protein populations

11 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

12 THE PRINCIPLE OF FRET A D
The probe D absorbes light at 430nm and emits light at 480nm The probe A absorbs at 480nm and emits at 545nm When these protein are brought in close proximity, energy transfer can occur (red row). 480 nm 430 nm 480 nm 545 nm Max 100 Å D A 545 nm EX. EM. EX. EM. 430 nm The probe D is excited by absorbing light at 430nm and transfers the energy to probe A. Now the probe A is excited and falls back to its ground state, thereby emitting light at 545nm. A D EM. EX. When D and A are in close proximity, the emission at 480 nm is decreased and the emission at 545nm is increased.

13 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

14 GFP Mutants …..what are the best FRET partners?

15 Primary conditions for FRET microscopy
Donor and Acceptor molecules must be in close proximity (tipically 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

16 Fluorescence resonance energy transfer
CFP Exc: 430 nm Em: 480 nm CFP Exc: 430 nm Em: 480 nm YFP Exc:475 nm Em: 545 nm

17 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.

18 Beamsplitter (2 Detectors)
FRET MICROSCOPE Beamsplitter (2 Detectors) sample objective Excitation CFP (430 nm) Dicroic (455 nm) Dicroic (505 nm) Emission YFP (545) Emission CFP (480nm) Detector YFP (FRET) CFP

19 FRET FRET CFP YFP CFP YFP YFP CFP
430 nm FRET CFP YFP The ratio of Donor to Acceptor emission has been used as an index of the extent of FRET FRET YFP CFP 430 nm 480nm 480nm 545nm 545nm

20 FRET APPLICATIONS: Variations in membrane potential Protease activity
Variations in intracellular Ca2+ and cAMP levels Conformational protein changes Protein-protein interactions

21 Cyclic AMP signalling From: G.M. Fimia and P. Sassone-Corsi, Journal of Cell Science 2001

22 Invasive signals from the microenvironment act as inducer of tumor progression
sIERO O 2

23 Cell migration in tumors
Collective motility (solid cell strands, sheets, files and clusters) Amoeboid motility (shape-driven migration path finding) Leading-edge rich in small pseudopodia Ellipsoid cell body Trailing uropodia Extrinsic factors from the microenvironment can promote tumor motility! Mesenchimal motility Surface protease path generation Direction of movement Leading-edge pseudopodia elongated and adhesive phenotipe

24 Normal, human breast cell line Human breast metastatic cell line
MCF10-A Normal, human breast cell line MDA-MB-435 Human breast metastatic cell line

25 Invasion specific Signal Transduction cascade
ROCK RhoA PKA P NHE1 cAMP Increased invasive capacity (+) Serum deprivation redistributes total RhoA, phospho RhoA, NHE1 in to MDA-MB-435 pseudopodial compartment

26 Are the mobilization of cAMP and activation of PKA localized to the leading edge pseusopodia of cancer cells? p38 ROCK RhoA Local pool of PKA? P NHE1 Local pool of cAMP? How this cAMP-mediated signalling is modulated by invasion promoting stimuli?

27 FRET FRET THE cAMP SENSOR IS A CHIMAERIC PROTEIN KINASE A 535nm 535nm
CAT R FRET FRET YFP YFP CFP CFP 480nm 480nm 430nm 430nm

28 GPCR AC g b ATP cAMP a CAT R Inactive PKA

29 GPCR AC g b a CAT CAT Active PKA R R

30 FRET How is it possible to measure cAMP with FRET? Low cAMP CAT YFP R
CFP CAT R FRET 430nm Low cAMP

31 YFP R CAT YFP CFP R CFP 430nm cAMP CAT Reg.
Zaccolo M. et al., Science ,2002.

32 cAMP sensors based on PKA
pBI-cRII-ycat RII CAT CFP YFP c pBI-RII-myrpalm RII CAT CFP YFP mp c

33 EPAC H30 myrpalm cytosolic HBE HBE Ponsioen et al.
EMBO Rep December; 5(12): 1176–1180 myrpalm cytosolic HBE HBE

34 A-Kinase activity reporter (AKAR)
Ni Q, Titov DV, Zhang J. Methods Nov;40(3): cytosolic myrpalm

35 Detection of local cAMP activity using FRET in both MCF10-A and MDA-MB-435 cells
EmCFP/EmYFP 0,9 1 1,15 MCF10 A mb psu ND D ** * *** MDA-MB-435 mb ND D ** psu 0,9 1 1,15 EmCFP/EmYFP RII-CFP EmCFP/EmYFP Low cAMP concentration High cAMP RII-CFP EmCFP/EmYFP High cAMP concentration Low cAMP concentration