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Fluorescence microscopy view of muscle
mechanochemistry
Dmitry Ushakov
Myosin, actin and striated muscle
Actin filament
Muscle actomyosin kinetic cycle
A.M M.ATP A.M.ADP.Pi A.M.ADP´A.M.ADP.Pi´ A.M.ADP´ A.MM.ADP.Pi
k1
r1
k2
r2
k3
r3
k4
r4
k5
r5
k6
r6
k7
r8A.M M.ATP A.M.ADP.Pi A.M.ADP´A.M.ADP.Pi´ A.M.ADP´ A.MM.ADP.Pi
k1
r1
k1
r1
k2
r2
k2
r2
k3
r3
k3
r3
k4
r4
k4
r4
k5
r5
k5
r5
k6
r6
k6
r6
k7
r8
k7
r8
Detachedstates
Three Force generatingstates
ADPATP
Weakly attached
Pi
Fluorescent Pi binding protein:
-Environmentally sensitive coumarin-based dye
-Pi binds tightly to the protein and increases coumarin fluorescence
Hirshberg M, Henrick K, Haire LL, Vasisht N, Brune M, Corrie JE, Webb MR. Biochemistry. 1998 37:10381-5.
Skinned fiber
Rigor (zero Ca2+, zero ATP)
Load Ca2+ and NPE-caged ATP
Load labelled sensor(PBP-MDCC)
Laser flash activation
Mechanical perturbation
ADP or Pi Dependent-Fluor
Forceproduction
Force and Pi release time courses
Time courses of force, sarcomere length, and phosphate release.
Experiments were at 12C. Fibers were activated from Ca21 rigor using a laser pulse to release ATP from NPE-caged ATP. Laser flash was at time 0, and the release step (0.5% of fiber length) was at 0.4 s (vertical dashed line in all records). Linear fits in panel C to the pre- (0.3–0.4 s) and post- (0.45–0.55 s) step data are shown by the thin lines.
Response to rapid length steps at 12C. (A) Phosphate release. Single exponentials, fitted through zero [Pi] at time 0.4 s, are fitted to the transients; rate constant are 35 s-1 (0.3%L, gray line) and 32 s-1 (0.5%L, black line). (B) Tension recovery records after rapid release steps of 0.3%L (gray) and 0.5%L (black).
Effect of temperature and ADP on cross-bridge dynamics
Calculated distribution of attached cross-bridges after rapid length steps. (A) Relative occupancy of AM’ADPPi (thin lines) and AM’ADP (thick lines) cross-bridge states during the period of a rapid release step, calculated by the model (Scheme 1). Calculations are shown for 20C with a 0.5%L step (black), 12C with a 0.5%L step (blue), and 12C with a 0.3%L (red). (B) Relative occupancy of AM’ADPPi (thin lines), AM’ADP (thick lines), and AMADP (dashed lines) cross-bridge states during the period of a 0.5%L step at 12C with (green lines) and without (blue lines) 1 mM added ADP. The AMADP state includes both AMADP (nonforce, ADP bound) and AM.cagedATP states.
A.M M.ATP A.M.ADP.Pi A.M.ADP´A.M.ADP.Pi´ A.M.ADP´ A.MM.ADP.Pi
k1
r1
k2
r2
k3
r3
k4
r4
k5
r5
k6
r6
k7
r8A.M M.ATP A.M.ADP.Pi A.M.ADP´A.M.ADP.Pi´ A.M.ADP´ A.MM.ADP.Pi
k1
r1
k1
r1
k2
r2
k2
r2
k3
r3
k3
r3
k4
r4
k4
r4
k5
r5
k5
r5
k6
r6
k6
r6
k7
r8
k7
r8
Brune, Corrie & Webb, 2001 Biochemistry 40:5087-5094
NDPK~P NDPK
ADP ATP
Nucleotide diphosphate kinase
The protocol for temperature jump (T-jump) activation:• fiber mounted in relaxing solution (5 mM ATP, zero Ca2+) at 0°C.• transfer to ‘pre-activating solution’ for 2 min (like relax, but replace EGTA with HDTA).• transfer to activating solution (5 mM ATP, 32 µM Ca2+, 1 mM Mg2+) for 2 seconds.• transfer to 12°C for 2 seconds
• either into a second activating solution (mechanics only).• or into silicone oil (mechanics + fluorescence)
• relaxation at 12°C.
ADP release in temperature jump (T-jump) activated fibres:• sensitive to small ΔADP (sub-micromole)• high time resolution
• sub millisecond response time• does not require the long equilibration time of the NADH method
• assay effects of rising Pi on mechanochemical coupling• obtain several contractions from a single fibre
-0.05
0.15
0.35
0.55
0.75
3700 3900 4100 4300
Fo
rce
(V)
0.5
0.6
0.7
0.8
0.9
1N
DP
K f
luo
resc
ence
(V
)
-0.05
0.15
0.35
0.55
0.75
3000 3500 4000 4500
Fo
rce
(V)
0.5
0.6
0.7
0.8
0.9
1
ND
PK
flu
ore
scen
ce (
V)
T-jump from 0 to 12°C
Shortening at 1.5 ML s-1
Time (ms)
[ADPt] = (Flt*Keq*[ATP+ADP]) / (Max. Fl + Flt(Keq – 1))
0.15 at 12ºC(West et al 2009)
Max Fl in fibre with:•60 M PNDPK-IDCC•50-100 M sulforhodamine•5 mM ATP vs 5mM ADP
0
100000
200000
300000
400000
500000
440 490 540 590 640 690Wavelength (nm)
Flu
ore
scen
ce (
AU
) 1.5 M sulforhodamine
1.0 M IDCC-NDPK~P
100 M ADP
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 0.5 1 1.5 2
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.5 1 1.5 2
Control (Po = 183.6 KN m-2)
+ 10 mM Pi (Po = 84.8 KN m-2)
Remove Pi (Po = 150.5 KN m-2)
P/Po
Velocity (ML s-1)
Velocity (ML s-1)
ADP release(mM s-1)
Shortening Speed (muscle lengths s-1)
0.0 0.5 1.0 1.5 2.0
AT
Pas
e ra
te (
mM
s-1
)
0.0
1.0
2.0
3.0
4.0
5.0
Pi release: ADP release:MDCC-PBP IDCC-NDPK
ADP release:IDCC-NDPK
0.31±0.03 mM s-1
T-jump activated
0.23±0.02 mM s-1
T-jump activated
10 mM Pi suppresses ATPase rate during shortening.• shape of ATPase-velocity relationship?• effects on force-velocity relationship, power velocity, efficiency?
ADP release and the effects of increased Pi during shortening
Isometric Force Control 195.0±7.7 KN m-2
+ 10 mM Pi 129.8±7.6 KN m-2
ADP release
P/Po0.0 0.2 0.4 0.6 0.8 1.0
Vel
ocity
(M
L s-1
)
0.0
0.5
1.0
1.5
2.0
0.0 0.5 1.0 1.5 2.0
0
5
10
15
20
25
30
35
40
Velocity (ML s-1)
Pow
er (
W)
Control
Vmax 2.15 ML s-1
Wmax 35 W
Vopt 0.62 ML s-1
Eff(Vopt) 0.37
a/Po 0.001 (0.2/195)
+ 10 mM Pi
1.63 ML s-1
37 W
0.68 ML s-1
0.51
0.01(1.14/129)
V = b (1 – P/Po)/(P/Po + a)
Eff = W/(GATP ATPase)
W = Po vel a (Vmax – vel)/(vel + b)
What happens to the lever arm?
Pre-power stroke state Post-power stroke state
The end of the lever arm moves about 11nm between the two states (Geeves & Homes, Annu. Rev. Biochem. 1999.68:687-728.)
Evidence of the lever arm rotationProtein crystallographyLow angle X-ray diffractionFluorescence polarisationFRETElectron microscopyEPR
Myosin essential light chain
• CaM-like EF-hand protein• Binds to a specific IQ-
sequence of myosin heavy chain
• Flexible structure – association equilibrium.
• Mammal isoform contains only one cysteine residue
Interface zone in the transition state
Highlighted are the residues within 5 Å of the opposite surface: Lys142, Gly143, Lys144, Glu148, Arg162, Gln166, Asp167, Arg168, Val258, Thr259, Tyr 261 of the heavy chain (yellow) and Lys97, Glu98, Met104, Ala106, Glu107, Arg109, His110, Thr114, Lys118, Glu125, Glu132, Ser134, Asn135 of ELC (green).
Locations of labelling sites and exchange of ELC into muscle fibers
ELC exchange:1. Incubation of skinned fibres in excess of labelled ELC at 370C in relaxing solution containing trifluoperazine (~70% ELC exchange).
2. Restoration of muscle by incubating with excess of Troponin C in relaxing solution at low temperature.
IDCC (coumarin)
Characterisation of ELC exchange
y = 0.2366x
R2 = 0.9885
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 5000 10000 15000 20000 25000 30000 35000 40000 45000
FluorescenceC
ou
mas
sie
Two-photon microscopyCo-localisation / confocal microscopy
Exchange efficiency
70% ELC exchange,50% labeling
Fiber activation
Fluorescence properties of ELC in solution
cys160
cys180
cys142
cys127
277
1700
1251
14161497
0
200
400
600
800
1000
1200
1400
1600
1800
IDCC LC127 LC142 LC160 LC180
Flu
ore
scen
ce l
ifet
ime,
ps
Fluorescence Lifetime Imaging
The isolated permeabilized muscle fibers were suspended on hooks in a trough chamber (bottom left) and incubated at 370C in exchange solution to introduce fluorescent ELC. The chamber was moved under upright Leica SP5 microscope equipped with a 63x/0.9 lens. The fluorescence was excited by a pulsed Mai Tai laser at 850 nm and the fluorescence lifetime images were recorded using a time-correlated single photon counting module. A typical fluorescence lifetime image with a lifetime distribution graph and a fluorescence decay from a single pixel are shown (top right). The force developed after fiber stretch was detected using a force transducer (bottom right).
Leica SP5 TCSPC63x/0.9
Fluorescence lifetime decay fitting
1. Open intensity image 2. Threshold 3. Create selection
4. Import lifetime matrix 5. Transfer selection from intensity image
6. Obtain lifetime distribution in selected area
Fluorescence lifetime of ELC in relaxed fibres
Difference between fluorescence lifetimes in relaxed fibers and in solutionELC lifetime distribution in relaxed fibers
Relation between lifetime and probe location
LC180 LC160
LC142LC127
277
1700
1251
14161497
0
200
400
600
800
1000
1200
1400
1600
1800
IDCC LC127 LC142 LC160 LC180
Flu
ore
scen
ce l
ifet
ime,
ps
Fluorescence lifetime of ELC in rigor fibres
Change of mean lifetime following relax to rigor transition
Fluorescence lifetime distributions of ELC180 in different fiber conditions
Response of lifetime to strainChange of mean lifetime following 1% stretch (F~150 kN/m2)
Half-maximal width of lifetime distributions
Proposed mechanism of actin binding and stretch effect on the fluorescence lifetime
Förster resonance energy transfer by FLIM
Rate constants for competing events:
D+A+hν
D*+A D+A*
D+A+hνD D+A+hνA
D+A D+A
kiD
kfD
kiA
kfA
kT
Donor
Acceptor
DEAC-ATP + IAF-ELC
FRET Couples CharacterizationDonor
MoleculeAlexa488 - ELC
Acceptor Molecule
Alexa594 – SH1
Donor Molecule
Alexa488 - ELC
Acceptor Molecule
Rhodamine - Actin
61
4230 107.9 JnQR D
dF
dFJ
D
AD4
72)488(
69.65)594488(
0
0
RhodamiAlexaR
AlexaAlexaR
6
0
1E
r1
R
ELC-SH1 FRET couple Characterization
Alexa488 - ELC
Alexa594 – SH1
Room Temperature Relaxing SolutionAlexa488-ELC + Alexa594-SH1
800ps
1900ps
5µm
Photons # Lifetime
Lifetime Measurements
Double Exponential Decay
Lifetime Measurements
Double Exponential Decay
Room Temperature Relaxing SolutionAlexa488-ELC + Alexa594-SH1
E=77±5%
800ps
1900ps
5µm
Photons # Lifetime
Non-Interacting DonorsD
Interacting DonorsDA
Phot
ons
Coun
t #
t [ns]
D
DAE
1
Num
ber
of O
ccur
renc
es
Lifetime MeasurementsAlexa488-ELC + Alexa594-SH1
E=70±5%
4
1
1800ps
2400ps
5µm
Photons # Chi2 – single exp Lifetime
Lifetime MeasurementsAlexa488-ELC + Alexa594-SH1
E=70±5%
4
1
1800ps
2400ps
5µm
Photons # Chi2 – single exp Lifetime
DA (ns) D (ns) E (%)
Alexa488-ELC + Alexa594-SH1
0.83± 0.21 2.20±0.12 63
0.78 ±0.12 2.24± 0.11 65
0.63±0.2 2.37±0.13 73
0.59±0.3 2.26±0.12 74
Alexa488-ELC 2.1±0.2
Alexa488-SH1 + Alexa594-ELC
0.49±0.16 2.29±0.15 80
0.45±0.13 2.45±0.08 82
0.47±0.16 2.29±0.14 79
0.49±0.16 2.46±0.18 80
Alexa488-SH1 2.6±0.2
Time (s)
For
ce (
kN m
-2)
Pi R
elea
se (
mM
)
force
Pi Release
Time-resolved fluorescence of Pi/ADP release and ELC in muscle fibers
Thank you!Laboratory of Muscle BiophysicsMichael FerencziValentina CaorsiTim WestDelisa Ibanez-GarciaAntonios KonitsiotisVerl Siththanandan (NIH, Bethesda)Marco Caremani (Florence)
Imperial Physics/PhotonicsPaul FrenchChris DunsbyHugh Manning
National Institute for Medical Research, London
Martin Webb
King’s College, LondonYin-Biao Sun