Molecular mechanisms of DNA repair and transcription studied by FRAP

Transcription

1 Molecular mechanisms of DNA repair and transcription studied by FRAP Adriaan Houtsmuller Josephine Nefkens Institute Erasmus Medical Centre Rotterdam The Netherlands

2 Nucleus GFP-tagged proteins

3

4

5 Confocal plane Focused laser beam

6 Confocal side view of a fixed cell expressing GFP

7 Confocal side view of a fixed cell expressing GFP

8 Confocal side view of a fixed cell expressing GFP

9 Confocal side view of a fixed cell expressing GFP

10 Spot bleaching of GFP in nuclei before bleach after bleach fixed immobile molecules living mobile molecules

11 Spot bleaching of GFP in nuclei before bleach after bleach fixed immobile molecules living mobile molecules

12 initiation: 1-2 minutes Single strand UV-damage XPC incision and synthesis: 3-5 minutes ERCC1/XPF XPA XPG POL I TFIIH AR GR transactivation: secon TIF2 SRC-1

13 pre-bleach post-bleach Spotbleaching of cells expressing ERCC1-GFP Spot-FRAP on ERCC1-GFP expressing cells before bleach FIXED after bleach fixed LIVING, NO UV untreated living 2 LIVING, UV (8 J/m ) UV irradiated (8 J/m 2 )

14 pre-bleach post-bleach Spotbleaching of cells expressing ERCC1-GFP Spot-FRAP on ERCC1-GFP expressing cells before bleach FIXED after bleach fixed LIVING, NO UV untreated living 2 LIVING, UV (8 J/m ) UV irradiated (8 J/m 2 )

15 pre-bleach post-bleach Spotbleaching of cells expressing ERCC1-GFP Spot-FRAP on ERCC1-GFP expressing cells before bleach FIXED after bleach fixed LIVING, NO UV untreated living 2 LIVING, UV (8 J/m ) UV irradiated (8 J/m 2 )

16 pre-bleach post-bleach Spotbleaching of cells expressing ERCC1-GFP Spot-FRAP on ERCC1-GFP expressing cells before bleach FIXED after bleach fixed LIVING, NO UV untreated living 2 LIVING, UV (8 J/m ) UV irradiated (8 J/m 2 )

17 pre-bleach post-bleach Spotbleaching of cells expressing ERCC1-GFP Spot-FRAP on ERCC1-GFP expressing cells before bleach FIXED after bleach fixed LIVING, NO UV untreated living 2 LIVING, UV (8 J/m ) UV irradiated (8 J/m 2 )

18 Spot-FRAP on ERCC1-GFP expressing cells FIXED fluorescence ratio profiles after/before n= LIVING, NO UV 0.8 after/before n= LIVING, UV (8 J/m ) after/before n=62 pre-bleach post-bleach distance to spot ( µ m)

19 Immobilisation of ERCC1-GFP depends on UV-dose 50 immobile fraction (%) (10,000) (300,000) (600,000) UV-C (J/m ) n=20 n=25 n=40 Houtsmuller et al., Science 1999

20 What if immobilisation is transient?

21 Strip-FRAP fast molecules relative fluorescence slow molecules Transiently immobile Immobile molecules fraction Time strip bleach pulse (0.1 s)

22 D=1 D=7 + transiently immobile

23 Combined FRAP and FLIP fluorescence loss in photobleaching (FLIP) (FRAP) fluorescence redistribution after photobleaching

24 D=1 D=7 + transiently immobile

25 D=4 D=7 + transiently immobile

26 D=4 D=7 + transiently immobile

27

28 COMPUTER SIMULATION OF FRAP ON FLUORESCENT NUCLEAR PROTEINS random motion stepsize D = stepsize 2 2*cycle time

29 COMPUTER SIMULATION OF FRAP ON FLUORESCENT NUCLEAR PROTEINS [immobile] immobilisation eq (e.g. P bind to = DNA-protein * average immobilisation time -1 complexes) [free] eq P release ~ t after binding ±σ

30 COMPUTER SIMULATION OF FRAP ON FLUORESCENT NUCLEAR PROTEINS bleaching P bleach = F(x,y,z) F ~ I laser (x,y,z) F ~ cycle duration

31 Computer simulation DEMO

32 A XPG TFIIH initiation: seconds AR GR transactivation: seconds TIF2 SRC-1 POL II elongation: minutes

33 Mobility of the Androgen Receptor (AR) studied by strip-frap and FLIP-FRAP

34 Mobility of an Androgen Receptor mutant that cannot bind DNA

35 Mobility of the Androgen Receptor (AR) and a non-dna-binding mutant Farla et al., J Struct Biol 2004

36 What if immobilisation is too short to be distinguished by FRAP?

37 A XPG TFIIH initiation: seconds AR GR transactivation: seconds TIF2 SRC-1 POL II elongation: minutes

38 1. Diffusion at different temperatures: Diffusion slows down linearly with temperature: D ~ T from 310 K (37 o C) to 300 K (27 o C) D will only go down ~3% (Phair and Misteli, 2000; Politz et al, 1998) 2. Immobilisation time at different temperatures: Proteins involved in a temperature dependent process will be immobilised much longer when temperature is dropped 10 degrees -> Overall mobility of proteins will only significantly decrease with temperature when they are transiently involved in a temperature dependent process in which they are immobilised (e.g. transcription)

39 Mobility of transcription factor TFIIH at different temperatures o C 37 o C Transcriptionally active Log (FRAP-FLIP) Time (s)

40 Mobility of transcription factor TFIIH at different temperatures o C 37 o C Transcriptionally active Log (FRAP-FLIP) 37 o C Transcription inhibited Time (s)

41 Mobility of transcription factor TFIIH at different temperatures o C 37 o C Transcriptionally active Log (FRAP-FLIP) 27 o C 37 o C Transcription inhibited Time (s) Hoogstraten et al., Mol Cell 2002

42 What if immobilisation is too long for FRAP?

43 initiation: 1-2 minutes XPC incision and synthesis: 3-5 minutes ERCC1/XPF XPA XPG POL I TFIIH initiation: seconds AR GR transactivation: se TIF2 SRC-1

44 Accumulation of TFIIH (XPB-GFP) in UV irradiated spots and in nucleoli

45 Fluorescence loss in photobleaching (FLIP) of TFIIH in a partly UV-irradiated cell before bleach after bleach equilibrium

46 FLIP of a cell expressing TFIIH-GFP before bleach after bleach equilibrium (300 s) UV irradiated Unirradiated Bleach spot relative fluorescence (I t /I 0 ) 0.3 bleach Time -30 (s) Time 270(s)

47 initiation: 1-2 minutes XPC incision and synthesis: 3-5 minutes ERCC1/XPF XPA XPG POL I TFIIH initiation: seconds AR GR transactivation: seconds TIF2 SRC-1 POL II elongation: 2-3 minutes elongation: minutes

48 initiation: 1-2 minutes XPC incision and synthesis: 3-5 minutes ERCC1/XPF XPA XPG POL I TFIIH initiation: seconds AR GR transactivation: seconds TIF2 SRC-1 POL II elongation: 2-3 minutes elongation: minutes

49 Repair and transcription factors move freely through the nucleus and have access to most regions of the nucleus Interaction of repair and transcription factors with DNA is dynamic allowing a rapid response to changing conditions; e.g. TFIIH can readily switch between transcription and repair Repair complexes are more stable than transcription initiation complexes TRANSCRIPTION transactivation by steroid receptors: seconds (~20%) initiation by TFIIH: 2-10 seconds (~30-60%) elongation: minutes DNA REPAIR initiation: 2-3 minutes (~40%) excision and synthesis: 3-5 minutes (~30%)

50 FRAP and FLIP are well suited to determine mobility parameters : -diffusion coefficient -immobile fraction -average duration of immobilisation Application is especially powerful in inducible systems like DNA repair and steroid receptor transcription initiation Computer simulation -understand and develop FRAP and FLIP assays -estimation of mobility parameters -simulate cellular processes and develop new assays to verify/falsify simulated models