observing molecular diffusion and binding with light sheet ... · observing molecular diffusion and...
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Observing moleculardiffusion and bindingwith light sheet FCSwith light sheet FCS
and point FRAP
First Irish/UK Light SheetFluorescence Microscopy Workshop
November 21, 2013
Malte WachsmuthCell Biology & Biophysics Unit
European Molecular Biology LaboratoryHeidelberg, Germany
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Molecular mobilities and interactions on the cellular level
Vital processes on a cellular level rely on• transport and diffusion• establishment and maintenance of
concentration gradients• distribution, accessibility, and occupation of
specific binding sites• specific interactions of molecules
The measurement of molecular mobilities and dynamics yields quantitatively
• biochemical parameters (dissociation constants, degrees of binding/multimerization, ion concentrations, pH)
• biophysical properties (diffusion coefficients, viscosities, connectivities of cellular compartments, elastic parameters)
• in a spatially resolved mannerBased on Spiller et al., Nature 465, 2010
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Morphogen gradients during development
MW 2013/11/21Gurdon & Bourillot, Nature 413 (2001)
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Imaging and measuring mobilities and interactions
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Wachsmuth et al., Biochim. Biophys. Acta 1783, 2008
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Mobility = diffusion + binding
impact of biological interactions
complex formation can result in:
• reduced real diffusion coefficient
g
reduced real diffusion coefficient
131,1K=∝ ββMW
D
• reduced apparent diffusion coefficient
⎟⎟⎠
⎞⎜⎜⎝
⎛+=
off
onapp 1
kkDD real
• transient and long-term immobilizationto be described as full reaction-diffusionscheme
⎠⎝ off
scheme
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Confocal fluorescence correlation spectroscopy (FCS)
laser
objective lens
ers
chro
icrr
ors
Single color FCS:
• concentrations c of fluorescent molecules
detectors
filt
dic
mi fluorescent molecules
• properties of diffusion/ transport processes
chro
ic
mirro
r
nhol
e
• diffusion coefficients D
dic mpi
G(τ)
1/N ∝ 1/c1/N ∝ 1/c
MW 2013/11/21τcorr ∝ 1/D
log τ
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FCS – counting single molecules
Diffusion induces Diffusion induces fluctuations of the number of molecules
N 3 N 4 N 2
I(t)
N = 3 N = 4 N = 2
<N> = 3
I(t)
This results in <I> fluctuations of the
fluorescence signal
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t
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FCS – autocorrelation analysis
δI(t)
0 t
G(τ)
( ) ( ) ( )I t I tG
δ δ + τ
l
( ) ( ) ( )( )O 2G
I tτ =
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log τ
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FCS – autocorrelation analysis
δI(t)
0 t
G(τ)
( ) ( ) ( )I t I tG
δ δ + τ
l
( ) ( ) ( )( )O 2G
I tτ =
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log τ
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FCS – autocorrelation analysis
δI(t)
0 t
G(τ)
( ) ( ) ( )I t I tG
δ δ + τ
l
( ) ( ) ( )( )O 2G
I tτ =
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log τ
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FCS – autocorrelation analysis
δI(t)
0 t
G(τ)
( ) ( ) ( )I t I tG
δ δ + τ
l
( ) ( ) ( )( )O 2G
I tτ =
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log τ
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FCS – autocorrelation analysis
G(τ)
1/N ∝ 1/c
log τ
τcorr ∝ 1/D
Fitting the autocorrelation function to appropriate model functions results in
• properties of the diffusion process
• the concentration
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• the concentration
of several species with different hydrodynamic properties
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Different species in the autocorrelation function
+ →←kon
+ ←koff
I(t) I(t)
G(τ)
t t
( )
t t
log τ
Properties of ligand-receptor interactions:
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dissociation constants, reaction rates, concentrations
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Confocal fluorescence cross-correlation spectroscopy (FCCS)
laser
objective lens
ers
chro
icrr
ors
Dual-color FCCS:
• bimolecular interaction properties
detectors
filt
dic
mi properties
• kinetic rates kon, koff
• dissociation constants KD
chro
ic
mirro
r
nhol
e
dic mpi
G(τ) concentrationinteractiondiffusion
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log τ
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FCCS – fluorescence cross correlation spectroscopy
E t d d tExtended concept:
• labeling of potential binding partners with spectrally different fluorophores
• looking for correlations between the corresponding signals
I(t) no correlationG(τ)
g p g g
( )
t log τ→ ←
kas
I(t) correlationG(τ)
→ ←
kdis
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t log τ
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FCCS – model application
→kon
+ →←koffoff
G(τ)
log τ
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FCCS – model application
→kon
+ →←koffoff
G(τ)
log τ
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FCCS – model application
→kon
+ →←koffoff
G(τ)
log τ
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Light-sheet FCCS
Jérémie CapouladeFriedhelm Serwane
Michael KnopTim SaundersLars Hufnagel
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Limitations of conventional and sequential point FCS
• Limited pool of (fluorescently labelled) molecules
• Heterogeneous cellular interior
• Slow cellular movements and changes
• Long integration time for 2D maps of FCS data
Single and sequential FCS measurements from
single cells often suffer from large errors
Biological requirements:
• good statistics in single cells
ti ll l d t
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• spatially resolved measurements
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Epi- vs. light-sheet fluorescence microscopy
Fluorescence excitation only in the detection focal plane• no out of focus fluorescence generated
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• optical sectioning deep inside 3D specimens • good S/N ratio
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Light-sheet-based imaging and multifocal FCS
2x Leica 40x/0.8NAwater dipping lens
illumination
detection
aqueousmedium
Huisken et al., Science 305, 2004
Light-sheet configuration:
Ill i ti & d t ti ith hi h NA t di i l• Illumination & detection with high NA water dipping lensesoptimized optical performance
• No agarose-based sample mountingit bl f li i llsuitable for living cells
• Combination with an inverted microscopesimplified sample positioning
MW 2013/11/21Capoulade et al., Nat. Biotechnol. 29, 2011
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2D-FCS: concept
Optical sectioning under 45°:
• Array of close-to-confocalvolume elements
EM-CCD-based detection:
• Single photon counting
• Every pixel serves as FCS point detector
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• Every volume element correspondsto a camera pixel
• Every pixel serves as FCS point detector
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2D-FCS: the setup
Intensityi iimaging
2D-FCSimagingimaging
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PSF measurements
Confocal microscope Leica SP52D-FCS microscopeλdet
Sample: 20nm beads (Invitrogen) in agarose
Lens: Lens: 63x, 1.2 NAλex Lenses:
Two 40x, 0.8 NA
λex λdetex det
(XY)(XY)
(XZ)(XZ)wXY = 370 nm
wZ = 410 nm
wXY = 240 nm
wZ = 600 nm(XZ)(XZ)V = 0.31 fL V = 0.19 fL
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Light-sheet microscopy of yeast with isotropic resolution
2D-FCS microscope
3D reconstructionimage stack, z step 280 nmscale bar 2.5 µm
scale bar 5 µm
confocal microscope
S. cerevisiae expressingplasma membraneprotein Pma1-GFP scale bar 5 µm
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p µ
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Light-sheet microscopy of Drosophila wing imaginal disc
Wing imaginal disc of Drosophila larva expressing GFP-NLS
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FCS of MDCK cells expressing the Fucci system
The fluorescent ubiquitination-based cell cycle indicator system Fucci:Sakaue-Sawano et al., Cell (2008)
• green in S to M phase
• red in G11
• in S to M, point FCS shows free diffusion of green construct with D ~ 24 µm2.s-1
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2D-FCS of MDCK cells expressing the Fucci system
QuantEMEM-CCD
S tiSensovationEM-CCD
2D-FCSmeasurements
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2D-FCS of GFP-NLS in Drosophila wing imaginal discs
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Chromatin organization in interphase
From histones to the chromatin fiber:• compaction• conservation and protection• conservation and protection• dynamic organisation• regulation of transcription,
replication repairreplication, repair• adoption of different epigenetic states
Wachsmuth et al., Biochim. Biophys. Acta 1783, 2008
Controversial:Controversial:• 30 nm chromatin fiber –
as observed under(semi-)dilute conditions(semi-)dilute conditions
• poorly structured –sea of nucleosomes, polymer melt
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Maeshima et al., Curr. Op. Cell. Biol. 22, 2010
polymer melt
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Chromatin organization in interphase
Models for higher-order organisation:
????Fractal globule model
Jhunjhunwala et al.,Cell 133, 2008
Mateos-Langerak et al.,PNAS 106, 2009
Lieberman-Aiden et al.,Science 326, 2009
Multi-loop sub-compartment model
Random-loop modelcompartment model
Verschure et al., J. Cell. Biol. 147, 1999Cook, Science 284, 1999
Meaburn & Misteli, Nature 445, 2007
Submicrometer-sized b h l l
Occupation of distincth
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subchromosomal replication and transcription foci
chromosome territoriesin the nucleus
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3T3 cells expressing HP1α-EGFP: diffusion and binding
Heterochromatin protein 1 isoform α (HP1α):
• involved in heterochromatin formation
bi d l b ll t h ti• binds globally to chromatin
• and with higher affinity to heterochromatin
Müller et al. (2009) Biophys. J. 97Cheutin et al. (2003) Science 299
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( ) p y( )
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Confocal FCS of 3T3 cells expressing HP1α-EGFP
2-component fit:
• 1st component is free fraction (fast diffusion)• 1st component is free fraction (fast diffusion)
• 2nd compoment is bound fraction (slow reaction-diffusion) with different properties in eu- and heterochromatin
However:
• overlap of eu- and heterochromatin distribution
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• overlap of eu- and heterochromatin distribution
• noise? significant? spatial distribution?
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2D-FCS of 3T3 cells expressing HP1α-EGFP
Intensity
1-comp. fit
Slow comp of 2 comp fitSlow comp. of 2-comp. fit
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2D-FCS of 3T3 cells expressing HP1α-EGFP
Intensity
1-comp. fit
Slow comp of 2 comp fitSlow comp. of 2-comp. fit
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2D-FCS of 3T3 cells expressing HP1α-EGFP
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Combined point FRAP and FCS
Kang-Bin ImUte Schmidt
Moonsik KangJi Young Lee
Felix Bestvater
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Fluorescence recovery after photobleaching (FRAP)
Re lt of FRAP e e i e tResults of FRAP experiments:
• properties of diffusion/transport processes
• diffusion coefficients D
• properties of interactions
• association dissociation rates • association, dissociation rates kon, koff
• relative concentrations of different fractionsdifferent fractions
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Combining FRAP and FCS
Comparing FRAP and FCS experiments:
• FCS and FRAP address different time, space and concentration regimes
• FCS allows precise determination of diffusion propertiesFCS allows precise determination of diffusion properties
• FRAP assesses properties of binding processes that involve immobilization
Limitations and drawbacks:
• unwanted diffusion during bleach step of FRAP sequence
imaging FRAP too slow to sample diffusion processes• imaging FRAP too slow to sample diffusion processes
• FCS is “conceptually blind” for immobilized molecules
• application of generalized diffusion-reaction models difficult due to the pp gheterogeneous cellular geometry and topology
• results for diffusion coefficients and transport parameters are often inconsistent
-> Combined point FRAP and FCS using a confocal microscope
-> Comprehensive theoretical description of FRAP and FCS
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> Comprehensive theoretical description of FRAP and FCS
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Confocal point FRAP and FCS
Modified confocal FCS microscope:Modified confocal FCS microscope:
• flexible AOTF control with 10 μs time resolution
• flexible APD readout with 1 μs time resolution
• flexible raw data processing• flexible raw data processing
-> Combined point FRAP and FCS measurements
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Point FRAP and FCS of EYFP in HeLa cells
HeLa cells expressing EYFP(scale bar 10 μm)
point FRAP with bleach times0.1, 0.5, 1, 5 ms
FCS
bleach timeT [ms]
half-time of recoveryτ1/2 [ms]
immobilized fraction
fimmo [%]
diffusion time τdiff [ms]
3D FRAP (Axelrod 1976)
3D FRAPwith diffusion
5 0 6 23 0 42 1 9 76 0 24 1 58 0 165.0 6.23 ± 0.42 < 1 9.76 ± 0.24 1.58 ± 0.16
1.0 2.23 ± 0.20 < 1 2.84 ± 0.08 0.72 ± 0.11
0.5 1.25 ± 0.17 < 1 1.22 ± 0.04 0.63 ± 0.12
0 3 1 03 ± 0 13 < 1 0 96 ± 0 04 0 63 ± 0 130.3 1.03 ± 0.13 < 1 0.96 ± 0.04 0.63 ± 0.13
0.1 0.78 ± 0.09 < 1 0.56 ± 0.02 0.45 ± 0.09
extrapolated to 0.0 0.78 ± 0.13 < 1 0.49 ± 0.18 0.50 ± 0.05
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global fit — — 1.61 ± 1.61 0.45 ± 0.05
FCS — — — 0.46 ± 0.07 D = 24 ± 3 μm2s-1
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Closed-form coupled reaction-diffusion model for FRAP
Ansatz:
• solution of differential equations including diffusion, association and dissociation
• approach similar to FCS theory established in Elson & Madge (1974)
• closed-form expression for FRAP intensity parametrized with reaction rates and diffusion coeff.
• validation with simulated data
parameter ranges:
kon = 2...2000 s-1
validation with simulated data
on
koff = 2...2000 s-1
D = 20 μm2s-1
w0 = 0 2 μmw0 0.2 μm
τdiff = 0.5 ms
Result:
• model is applicable to a wide range of reaction vs • model is applicable to a wide range of reaction vs. diffusion rates
• coverage of (fully) coupled and (apparently) uncoupled reaction-diffusion schemes
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Dynamics of the exon-exon junction complex
(Schmidt et al., RNA, 2009)
Exon-exon junction complex (EJC):
• forms upon intron excision on maturating mRNA molecules in the nucleus
• serves as adaptor for nuclear-cytoplasmic export and for mRNP quality control • serves as adaptor for nuclear cytoplasmic export and for mRNP quality control
• core-shell model of the EJC: core formed from proteins binding after, shell formed from proteins binding prior to intron excision
• strong nuclear localization with accumulation in splicing specklesstrong nuclear localization with accumulation in splicing speckles
• highly dynamic and mobile
What contributes to the mobilities of the EJC components Magoh and REF2 II?
MW 2013/11/21
What contributes to the mobilities of the EJC components Magoh and REF2-II?
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Point FRAP and FCS of EJC factors REF2-II and Magoh
REF2-II-EGFPscale bar 5 μm
Magoh-EGFP
MW 2013/11/21
scale bar 5 μm
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Binding and diffusion properties of REF2-II and Magoh
FRAP FCSfa
st
mp.
f c
omp.diff
. co
m
wly
diff
usiv
e
ns.
boun
d
slow
tran
mob
.im
m
Im et al., Cytometry A (in press)
MW 2013/11/21