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UNIVERSITA ’ DEGLI STUDI DI BARI FACOLTA ’ DI SCIENZE MATEMATICHE FISICHE E NATURALI Studio dei segnali di trasduzione in cellule in vivo: applicazione.

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Presentazione sul tema: "UNIVERSITA ’ DEGLI STUDI DI BARI FACOLTA ’ DI SCIENZE MATEMATICHE FISICHE E NATURALI Studio dei segnali di trasduzione in cellule in vivo: applicazione."— Transcript della presentazione:

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 Fluorescence Process 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. Stage 1: Excitation Stage 2: Excited-State Lifetime Stage 3: Fluorescence Emission

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

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

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

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

7 GFP is from the chemiluminescent jellyfish Aequorea victoria

8 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 G reen F luorescent P rotein GFP

9 Fluorescent protein spectral variants 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

12 430 nm 545 nm 430 nm 480 nm D A EX.EM. EX. EM. EX. THE PRINCIPLE OF FRET 1)The probe D absorbes light at 430nm and emits light at 480nm 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). 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. When D and A are in close proximity, the emission at 480 nm is decreased and the emission at 545nm is increased. Max 100 Å A D

13 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 FRET

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

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 YFP Exc:475 nm Em: 545 nm CFP Exc: 430 nm Em: 480 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 sample objective Excitation CFP (430 nm) Dicroic (455 nm) Dicroic (505 nm) Emission YFP (545) Emission CFP (480nm) Detector YFP (FRET) Detector CFP Beamsplitter (2 Detectors) FRET MICROSCOPE

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

20 FRET APPLICATIONS:  Variations in membrane potential  Protease activity  Variations in intracellular Ca 2+ 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 sIEROsIERO

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 Mesenchimal motility Surface protease path generation Direction of movement Leading-edge pseudopodia elongated and adhesive phenotipe Extrinsic factors from the microenvironment can promote tumor motility!

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

25 Invasion specific Signal Transduction cascade Serum deprivation redistributes total RhoA, phospho RhoA, NHE1 in to MDA-MB-435 pseudopodial compartment p38 ROCK RhoA PKA P NHE1 cAMP Increased invasive capacity (+)

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

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

28 Inactive PKA    AC GPCR CAT RR ATP cAMP

29    AC GPCR Active PKA CAT RR

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

31 cAMP YFP CFP YFP CFP CA T RR 430nm YFP CAT YFP CAT CFP R Reg. R CF P Zaccolo M. et al., Science,2002.

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

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

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

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


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