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