Transcript Strain sensitivity of the myosin essential light chain in

Fluorescence microscopy
view of muscle
mechanochemistry
Dmitry Ushakov
Myosin, actin and striated muscle
Actin filament
Muscle actomyosin kinetic cycle
Detached
states
k1
A.M
k2
M.ATP
r1
r2
ATP
Weakly
attached
k3
M.ADP.Pi
r3
Three Force generating
states
k4
A.M.ADP.Pi
r4
A.M.ADP.Pi´
k5
k6
A.M.ADP´
r5
k7
A.M.ADP´
r6
Pi
ADP
A.M
r8
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
ADP or Pi
Dependent
D-Fluor
Force
production
Mechanical perturbation
Force and Pi release time courses
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).
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.
Effect of temperature and ADP on
cross-bridge dynamics
k1
A.M
k2
M.ATP
r1
r2
k3
M.ADP.Pi
r3
k4
A.M.ADP.Pi
r4
A.M.ADP.Pi´
k5
k6
A.M.ADP´
r5
k7
A.M.ADP´
r6
A.M
r8
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.
Nucleotide diphosphate kinase
NDPK~P
ADP
Brune, Corrie & Webb, 2001 Biochemistry 40:5087-5094
NDPK
ATP
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
[ADPt] = (DFlt*Keq*[ATP+ADP]) / (Max. Fl + DFlt(Keq – 1))
0.15 at 12ºC
(West et al 2009)
T-jump from 0 to 12°C
0.9
0.8
0.35
0.7
0.15
-0.05
3000
0.6
3500
4000
4500
0.5
1
0.75
0.9
Force (V)
0.55
0.8
0.35
0.7
0.15
-0.05
3700
Max DFl in fibre with:
•60 mM PNDPK-IDCC
•50-100 mM sulforhodamine
•5 mM ATP vs 5mM ADP
0.6
3900
4100
Time (ms)
0.5
4300
Fluorescence (AU)
Force (V)
0.55
NDPK fluorescence (V)
Shortening at 1.5 ML s-1
NDPK fluorescence (V)
0.75
1
1.5 mM sulforhodamine
1.0 mM IDCC-NDPK~P
100 mM ADP
500000
400000
300000
200000
100000
0
440
490
540
590
Wavelength (nm)
640
690
P/Po
0.8
0.7
Control
0.6
+ 10 mM Pi (Po = 84.8 KN m-2)
0.5
(Po = 183.6 KN m-2)
Remove Pi (Po = 150.5 KN m-2)
0.4
0.3
0.2
0.1
0
0
0.5
1
1.5
-1)
Velocity
(ML
s
2
ADP release
4
(mM s-1)
3.5
3
2.5
2
1.5
1
0.5
0
0
0.5
1
1.5
2
Velocity (ML s-1)
ADP release and the effects of increased Pi during shortening
Pi release:
MDCC-PBP
ADP release:
IDCC-NDPK
-1
ATPase rate (mM s )
5.0
ADP release
0.31±0.03 mM s-1
T-jump activated
4.0
Control
3.0
+ 10 mM Pi 129.8±7.6 KN m-2
2.0
ADP release:
IDCC-NDPK
1.0
0.0
s-1
0.23±0.02 mM
T-jump activated
Isometric Force
195.0±7.7 KN m-2
0.0
0.5
1.0
1.5
2.0
Shortening Speed (muscle lengths s-1)
10 mM Pi suppresses ATPase rate during shortening.
• shape of ATPase-velocity relationship?
• effects on force-velocity relationship, power velocity, efficiency?
2.0
V = b  (1 – P/Po)/(P/Po + a)
W = Po  vel  a (Vmax – vel)/(vel + b)
-1
Velocity (ML s )
1.5
1.0
Eff = W/(DGATP  ATPase)
0.5
0.0
0.0
0.2
0.4
0.6
0.8
1.0
P/Po
40
35
Vmax
30
Power (W)
25
Wmax
Control
+ 10 mM Pi
2.15 ML s-1
1.63 ML s-1
35 W
37 W
20
Vopt
15
0.62 ML s-1 0.68 ML s-1
Eff(Vopt) 0.37
10
5
a/Po
0
0.0
0.5
1.0
1.5
-1
Velocity (ML s )
2.0
0.001
(0.2/195)
0.51
0.01
(1.14/129)
What happens to the lever arm?
Evidence of the lever arm rotation
Protein crystallography
Low angle X-ray diffraction
Fluorescence polarisation
FRET
Electron microscopy
EPR
Pre-power stroke state
Post-power stroke state
The end of the lever arm moves about 11
nm between the two states
(Geeves & Homes, Annu. Rev. Biochem. 1999.68:687-728.)
Myosin essential light chain
• CaM-like EF-hand protein
• Binds to a specific IQsequence 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).
IDCC (coumarin)
2. Restoration of muscle by incubating with
excess of Troponin C in relaxing solution at
low temperature.
Characterisation of ELC exchange
Two-photon microscopy
Co-localisation / confocal microscopy
Exchange efficiency
10000
y = 0.2366x
R2 = 0.9885
9000
8000
Coumassie
7000
6000
5000
4000
3000
2000
70% ELC exchange,
50% labeling
Fiber activation
1000
0
0
5000
10000
15000
20000
25000
Fluorescence
30000
35000
40000
45000
Fluorescence properties of ELC in
solution
cys180
cys160
cys142
cys127
1800
1700
1600
1497
Fluorescence lifetime, ps
1416
1400
1251
1200
1000
800
600
400
277
200
0
IDCC
LC127
LC142
LC160
LC180
Fluorescence Lifetime Imaging
Leica SP5 TCSPC
63x/0.9
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).
Fluorescence lifetime decay fitting
1. Open intensity image
4. Import lifetime matrix
2. Threshold
5. Transfer selection
from intensity image
3. Create selection
6. Obtain lifetime distribution
in selected area
Fluorescence lifetime of ELC in
relaxed fibres
ELC lifetime distribution in relaxed fibers
Difference between fluorescence
lifetimes in relaxed fibers and in solution
Relation between lifetime and
probe location
LC180
LC160
1800
1700
1600
1497
Fluorescence lifetime, ps
1416
1400
1251
1200
1000
800
600
400
277
200
0
IDCC
LC142
LC127
LC142
LC160
LC180
LC127
Fluorescence lifetime of ELC in
rigor fibres
Fluorescence lifetime distributions of
ELC180 in different fiber conditions
Change of mean lifetime following relax to
rigor transition
Response of lifetime to strain
Change 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
Donor
Acceptor
Rate constants for competing events:
D+A
D+A+hν
kiD
D*+A
D+A
kT
kiA
D+A*
kfD
kfA
D+A+hνD
D+A+hνA
DEAC-ATP + IAF-ELC
FRET Couples Characterization
Donor
Molecule
Alexa488 ELC


R0  9.7 10 3  2QD n 4 J  
1
6
 F     d
J   
 F  d
4
D
A
D
Acceptor
Molecule
Alexa594 –
SH1

R0 ( Alexa488 Alexa594)  65.69
Acceptor
Molecule
Rhodamine Actin

R0 ( Alexa488 Rhodam)i  72
E
1
 r 
1 

 R0 
6
Donor
Molecule
Alexa488 ELC
ELC-SH1 FRET couple Characterization
Alexa488 ELC
Alexa594 –
SH1
Lifetime Measurements
Alexa488-ELC + Alexa594-SH1
Room TemperatureRelaxing Solution
Photons #
1900ps
Lifetime
5µm
800ps
Double Exponential Decay
Lifetime Measurements
Alexa488-ELC + Alexa594-SH1
Room TemperatureRelaxing Solution
Lifetime
Number of Occurrences
Photons #
1900ps
E=77±5%
5µm
800ps
Photons Count #
Double Exponential Decay
Interacting Donors
DA
E  1
Non-Interacting Donors
D
t [ns]
 DA
D
Lifetime Measurements
Alexa488-ELC + Alexa594-SH1
1
Photons #
Chi2 – single exp
2400ps
Lifetime
5µm
4
1800ps
E=70±5%
Lifetime Measurements
Alexa488-ELC + Alexa594-SH1
1
Photons #
Chi2 – single exp
2400ps
Lifetime
5µm
4
1800ps
Alexa488-ELC + Alexa594SH1
E=70±5%
DA (ns)
D (ns)
E (%)
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
Alexa488-SH1 + Alexa594ELC
Alexa488-SH1
2.1±0.2
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
2.6±0.2
Time-resolved fluorescence of Pi/ADP
release and ELC in muscle fibers
Pi Release (mM)
Force (kN m-2)
force
Pi Release
Time (s)
Thank you!
Laboratory of Muscle Biophysics
Michael Ferenczi
Valentina Caorsi
Tim West
Delisa Ibanez-Garcia
Antonios Konitsiotis
Verl Siththanandan (NIH, Bethesda)
Marco Caremani (Florence)
Imperial Physics/Photonics
Paul French
Chris Dunsby
Hugh Manning
National Institute for Medical
Research, London
Martin Webb
King’s College, London
Yin-Biao Sun