Fluorescence fluctuation microscopy: FRAP and FCS

Transcription

1 Fluorescence fluctuation microscopy: FRAP and FCS

2 Confocal laser scanning fluorescence microscope for the mobility analysis in living cells

3 Ensemble measurements of protein mobility and interactions pre post: s 1 s 6 s spatial/temporal resolution bleach once FRAP intensity FRAP spatial analysis bleach intensity width 2 time 1 µm 1 ms - hours 1 µm 1 ms - hours continue bleach CP/ FLIP bleach: s 1 s 6 s intensity time 1 µm 1 ms - hours time correlate.3 µm 1 µs - 1 s Wachsmuth, M., Caudron-Herger, M. and Rippe, K. (28). Biochim. Biophys. Acta 1783,

4 Methods comparison FRAP (for slow/immobile particles, ~1 ms time resolution) - diffusion coefficients - rate constants (at immobile substrates) - immobile fractions - photoactivation instead of bleaching (koff measurements) CP/FLIP (for faster particles) - dissociation rate (at immobile substrates) FCS (for fast particles, µs time resolution) - diffusion coefficients - rate constants (at mobile substrates) - anomaly parameters - concentrations

5 The problem: many things affect the observed mobility collision with immobile chromatin heterogenous binding sites active, directed transport complex formation homgenous binding sites caging

6 Protein mobility and interactions in the cell freely mobile trajectory free time time f et acil lo ita ca te tio d n rg or an gan d iza bi ti nd on in g lf- ta co m pl confined/ moving corral se anomalous ex an for d ma bi ti nd on in g immobilízed Mean Squared Displacehment (MSD) MSD = 6 D t α Dependence of diffusion coefficient D and molecular mass M protein: D M 1 3 double mass M =>.8 fold lower D DNA: D M 1 2 double mass M =>.7 fold lower D Wachsmuth, M., Caudron-Herger, M. and Rippe, K. (28). Biochim. Biophys. Acta 1783,

7 Determining diffusion coefficient D, kinetic binding rates kon and koff, and the apparent equilibirium constant Keq * diffusion without binding, α = 1 for free diffusion transient chromatin binding strong chromatin binding D eff k on k off r D, α D free r 2 = 6 D t α D eff = D * K 1+ K * eq = k * on = k S on eq k off [ ] eq k off Erdel, Müller-Ott, Baum, Wachsmuth & Rippe (211) Chromosome Res 19,

8 Pericentric heterochromatin (PCH) in mouse cells telomere telomere euchromatin centromere (minor satellites) pericentric heterochromatin (PCH) (major satellites repeats) Mouse pericentric heterochromatin - chromocenters visualized by DAPI staining - 5meC, H3K9me3, H4K2me3 - HP1, Suv39h, Suv4-2h - silences transcription of repeats (requires Suv39h) - important for chromosome segregation 5 µm Specificity, propagation and epigenetic memory?

9 Writing, reading and transmitting epigenetic signals pioneering factor writer reader spreading of modification? histone modification from Molecular Biology of the Cell

10 Distinct chromatin states can be established and maintained via interlinked feedback loops silenced H3K9me3 heterochromatin H3K9me3 histone H3 lysine 9 methylation DNA methylase DNMT histone methylase histone acetylase HP1 SUV39h HAT SUV39h, DNMT Jmjd2 HAT HDAC histone H3 lysine 9 acetylation 5meC MeCP1 HDAC histone deacetylase histone demethylase H3K9ac active H3K9ac euchromatin

11 Colocalization of heterochromatin protein 1 (HP1), Suv39H1 histone methylase and H3K9 methylation DAPI GFP-HP1A H3K9me3-Alexa568 Merge GFP-HP1A TagRFP-Suv39H1 Merge pericentric heterochromatin DAPI 1 µm

12 Fluorescence Recovery After Photobleaching (FRAP)

13 Fluorescence Recovery After Photobleaching (FRAP) Protocol: 1. Bleach particles (with a laser) 2. Wait and watch during they diffuse away 3. Fit the fluorescence-over-time curve or profile Source: Wikipedia

14 example: ER marker protein Reits et al. 21 (From fixed to FRAP: measuring protein mobility and activity in living cells)

15 mobile protein immobile protein

16 Typical FRAP curve F i : f.i. before bleaching F : f.i. just after bleaching F : f.i. in recovery region mobile fraction Equation for the recovery curve in the absence of binding: characteristic diffusion time bleach radius w diffusion coefficient D τ D = w 2 4D F i (t) = e 2τ D 2τ t I D 2τ + I D t 1 t I, I 1 : Modified Bessel functions

17 FRAP with fast binding Fast binding: Reduction of the diffusion coefficient, shape of the curve remains unchanged Fast means: Many binding events occur during translocations on the length scale of the bleach spot Only one fit parameter: Effective diffusion coefficient D eff = D 1 + k * on k off If D is known, the ratio of the rate constants is obtained

18 FRAP with slow binding Long-lived binding events lead to different shape of recovery curve Data slow binding Effective diffusion (no or fast binding):.8.6 free/effective diffusion F i (t) = e 2τ D 2τ t I D t Slow binding: 2τ + I D 1 t.4 F i (t) =1 e k off t time (s) dissociation rate Moderately fast binding: no analytical solution

19 Intensity analysis FRAP resolves HP1 diffusion and interactions on the 1 µm and second time scale immobilized fraction (~ 1%) only in heterochromatin residuals relative intensity diffusion model reaction model diffusion-reaction model time (s) time (s) time (s) diffusion-reaction analysis (Sprague & McNally 24, Biophys. J. 86, 3473) yields k off =.15 ±.7 s -1 in heterochromatin

20 FRAP profile analysis yields an effective nuclear diffusion coefficient of Deff = 1.4 µm² s -1 of HP1 (1 µm and second scale) fit to a confined diffusion model

21 Diffusion versus binding in FRAP profile analysis diffusion dominant case y position 5 [mm] -5 1 relative.5 intensity -5 postbleach time s 13.3 s s 5 5 x position [mm] s binding dominant case y position 5 [mm] -5 1 relative.5 intensity -5 postbleach time s 13.3 s x position [mm] 43.3 s s Calculations Malte Wachsmuth

22 Mobility and interaction analysis in living cells Fluorescence recovery after photobleaching (FRAP) intensity 5 µm prebleach postbleach.3 s postbleach 4 s time Fluorescence correlation spectroscopy (FCS) intensity time (µs) correlation fct. log (time) Müller et al. (29). Biophys. J. 97, ; Erdel et al. (211) Chromosome Res 19,

23 Fluorescence correlation spectroscopy

24 The concept: measuring number fluctuations of fluorescent particles in the focus Diffusion induces fluctuations of the number of molecules N = 3 N = 2 N = 4 <N> = 3 F(t) <F> F(t) F(t) F This results in fluctuations of the fluorescence signal t

25 Fluctuations of the particle number of a 1 nm rhodamine solution in dependence of the observation volume Size [mm] Volume [l] particles N N/N [%]

26 FCS data analysis F(t) <F> We want to know: the average number of molecules in the focus concentration the dwell time in the focus diffusion coefficient

27 The concept: autocorrelation analysis δf(t) t τ G(τ) = 2 log τ

28 The concept: autocorrelation analysis δf(t) t τ G(τ) = 2 log τ

29 The concept: autocorrelation analysis δf(t) t τ G(τ) = 2 log τ

30 The concept: autocorrelation analysis δf(t) t τ G(τ) = 2 log τ

31 Results from FCS experiments G(τ) G( ) F( t) F( t ) F( t) 2 1/N 1/c τ corr 1/D log τ variance of fluctuations concentration length of fluctuations diffusion coefficient

32 Theoretical approach - formulas Properties of the optical system Properties of the diffusion process = I (r) =... c (r,t) =... G(τ) Analytical autocorrelation function concentration, brightness, diffusion properties of particles, interactions, cellular environment... log τ

33 FCS correlation function for free diffusion in 3D The correlation function G(τ) gives the probability to detect a particle at time t and at time t + τ That s how a point in the confocal microscope looks like detection efficiency t=1 That s how a diffusing molecule spreads out over time (in 1 dimension) t=2 Green s function for free 3D diffusion

34 FCS correlation function for free diffusion in 3D Probability to detect a particle before and after it has diffused for time τ definition of the correlation function correlation function diffusion time, concentration, focal volume, structure parameter, focus radius G(τ) τ diff resulting function log τ

35 Determining diffusion coefficients small molecules generate short fluctuations... F(t) larger complexes generate longer fluctuations... F(t) t t G(τ)... and rapidly decaying correlation functions log τ... and slowly decaying correlation functions

36 Measuring ligand binding affinity + kon k off I(t) I(t) G(τ) t t log τ Properties of ligand-receptor interactions: dissociation constant, reaction rates, concentrations

37 Fluorescence correlation spectroscopy (FCS) of the H3K9me3 histone methylase Suv39h1 FCS measurements at different locations in the cell G(τ) normalized cytoplasm D = 17 µm² s -1 euchromatin heterochromatin τ (µs)

38 Pixel-wise Photobleaching Profile Evolution Analysis - 3PEA bleach image intensity distribution during scanning acquisition low mobility low mobility 1. bleach line t = 3 τ l 2. bleach line t = 4 τ l 3. bleach line t = 5 τ l high mobility high mobility Erdel & Rippe (212). PNAS 19, E

39 3PEA analysis of Snf2H chromatin remodeler: Deff = 6.5 µm2 s-1 (FRAP:.7 µm2 s-1) RFP Merge experiment Snf2H-GFP D = 44 µm2s-1 fit result D = 6.5 µm2s Snf2H-GFP RFP SSR D (µm2s-1) D (µm2s-1) D (µm2s-1) 8 1

40 camera (area) Wide-field verus confocal microscopy setup fluorescent probe objective reconstruction of the spatial fluorescence distribution laser objective probe synchronisation of scan mirror and signal acquisition light source detector (point) filters pinhole dichroic mirror rotating scan mirror

41 Dynamic processes in the cell nucleus take place on the microsecond to hour time scale molecular diffusion molecular interactions processes particle/structural dynamics fluctuation spectroscopy photobleaching methods methods imaging/tracking 1 µs 1 ms 1 s 1 s Wachsmuth, M., Caudron-Herger, M. and Rippe, K. (28). Biochim. Biophys. Acta 1783,

42 typical resolution acquisition rate (frames/sec) light exposure wide field 25 nm (x,y) > 2 µm (z) 2 low confocal 25 nm (x,y) 6 nm (z) 1-1 high line scanner confocal 25 nm (x) 38 nm (y) 7 nm (z) 3 low

43 Typical values for diffusion coefficients in the nucleus Dmin (µm 2 s -1 ) accesible corral radius methods chromatin/ telomeres µm.2 µm.3-.8 µm CLSM, single particle tracking transcription factor 1-15 (free) -.1 up to 1 µm (nucleus) FRAP (bound) FCS (free, transiently bound) membrane proteins 2-2 (2-D) 1 µm (nuclear membrane) FRAP FCS

44 Accessible range of diffusion coefficients for CLSM, FRAP and FCS measurments tmin / tmax typical analysis volume Dmin / Dmax confocal (single particle tracking).4-2 sec / infinite 1 x 1 µm (x,y) 6 nm (z) µm 2 s -1 / 1 µm 2 s -1 FRAP.4-2 sec / infinite 2 x 2 µm (x,y).6-5 µm (z) µm 2 s -1 / 1 µm 2 s -1 FCS 1 µsec / 1 sec 25 nm (x,y) 6 nm (z).5 µm 2 s -1 / 2 µm 2 s -1