University APS Workshop on Biology of Pennsylvania March ... · APS Workshop on Biology March 12,...

Universityof

Pennsylvania

Universityof

Pennsylvania

Universityof

Pennsylvania

• Molecular Motors

• Muscle Energetics and Strain Dependence

• Unconventional Myosins – Myosin V

• Single-Molecule Fluorescence Polarization

• Fluorescence Imaging at 1 – 2 nm

• Defocussed Orientation and Positional Imaging

• Challenges in Molecular Motor Research

APS Workshop on Biology March 12, 2006

[email protected]

(Classic) Molecular Motors

Helicase Unwinds DNA Ribosome Synthesizes Proteins

Medalia et al. 2002 Science. 298:1209-13.

Artist’s ImaginationCryo-EM of Cell

David C. Goodsell

from Rhoades & Pflanzer, Human Physiology

Sarcomere Structure

2

Deterministic Tilting Motion

3

4

1The Actin-Myosin Cycle

0 0.2 0.4 0.6 0.8 10

0.5

1

1.5

2

Relative Force

Velocity ( m / s)

0 0.2 0.4 0.6 0.8 10

0.1

0.2

0.3

Relative Force

Power Relative to P Vµ o max

PennPennPennForce-Velocity, Power and Energy Utilization

Fenn and Hill, J. Physiol. 1932

0 0.2 0.4 0.6 0.8 10

0.5

1

1.5

2

Relative Force

Velocity ( m / s)µ

0 0.2 0.4 0.6 0.8 10

0.1

0.2

0.3

Relative Force

Power Relative to P Vo max


W●

H●

W ●

+

W = Power = F dx/dt = F · V●


Force-Velocity, Power and Energy Utilization

0 0.2 0.4 0.6 0.8 10

0.5

1

1.5

2

Relative Force

Velocity ( m / s)µ

0 0.2 0.4 0.6 0.8 10

0.1

0.2

0.3

Relative Force


PennPennPenn

W●

H●

W ●

+



0 0.2 0.4 0.6 0.8 10

0.5

1

1.5

2

Relative Force

Velocity ( m / s)µ

0 0.2 0.4 0.6 0.8 10

0.1

0.2

0.3

Relative Force



W●

H●

W ●

+



Isometric Three-Bead Assay

Barbed-End Pointed-End

Actin Filament

Motor Trapand Bead

Transducer Trapand Bead Myosin

Pedestal

B

A

Force = ~10 picoNewtons

∆GATP= ~100 zepto Joules

Displacement = ~10 nanometers

W = ½ F · D= ~50 x 10-21 Joules= ~50 zepto Joules

Universityof

Pennsylvania

Universityof

Pennsylvania

Universityof

Pennsylvania

Vesicle Movement in Cell ExtractNira Pollack & Ron D. Vale, UCSFFrom: Molecular Biology of the Cell, 4th ed.

Melanosome MovementJohn A. Hammer, III, NIH

Molecular Motors in Non-Muscle Cells

Myosin Family Tree

Myosin V Processivity

20,000 nm10x speed

Myosin V

Stalk

Cargo BindingDomain

Calmodulins

Motor Domains

Actin Filament 37 nanometers

Myosin V

Stalk

Cargo BindingDomain

Calmodulins

Motor Domains

Actin Filament Monkey Bars

Myosin V

Stalk

Cargo BindingDomain

Calmodulins

Motor Domains

Actin Filament Monkey Bars37 nanometers

B

C

D

A

Hand-Over-Hand Model of Processive Transport

Bifunctionally Labeled Calmodulin on Myosin V

Cys66

Cys73

Calmodulin Motor Domain

Tail

Calmodulins

Cargo BindingDomain

Stoichoimetry, Specificity and Cross-linking:• HPLC• Mass spectrometry • Tryptic digestion

Bifunctional Rhodamine on MyosinRegulatory Light Chain

bwiggle.avi

laser in laser outθ

φ(-X)

Z

Y

quartz coupling element

quartz slideevanescentwave

MM

laser

OL

QCE

L

RM

BF

ICCD

PPC

pPS

sX

Z

Y

APDs

SFLL

L

BSPX

Y

L

path 1path

2

Experimental Apparatus

Joe Forkey

Experimental Apparatus

MM

laser

OL

QCE

L

RM

BF

ICCD

PPC

pPS

sX

Z

Y

APDs

SFLL

L

BSPX

Y

L

path 1path

2s2yp2y

s1xp1x

s1yp1y

s2xp2x

Pol

ariz

ed In

tens

ity

time

Margot Quinlan

0

10 0

20 0

30 0

0

50 0

1 00 0

0

3 0

6 0

9 0

3 0

6 0

9 0

s2yp2y

s1xp1x

s1yp1y

s2xp2x

WeightedTotal

Myosin V - 5µM ATP

Pol

ariz

edFl

uore

scen

ce C

ount

sTo

tal

Inte

nist

y

1

time (s)53 420 1

Probeorientationdistribution

Photoselectedorientation

distributions

Overallorientationdistribution

H

V

Excitation


distributions

Observed through emission polarizers

Overallorientationdistribution

HIV

V

H

V

V

H

Excitation Detection

HIV

VIH

VIV

H


distributions

Observed through emission polarizersOverall

orientationdistribution

HIH

V

H

V

V

H


HIV

VIH

VIV

H


distributions

Observed through emission polarizersOverall

orientationdistribution

HIH

V

H

V

V

H


HIV

VIH

VIV

H

Emission polarization ratios

not Equal

114

=VIH

VIV

26

=VIH

VIV

H

Photoexcitedmolecule

Observed through emission polarizersSingle static

molecule

V

H

V

V

H


Equal

HIH

HIV

VIH

VIV

13

=VIH

VIV

26

=VIH

VIV


H

Orientation distributions of

several excitations

Observed through emission polarizers

4 ns << τ << 10 ms V

H

Excitation

V

V

H

Detection


not Equal

Single slowly wobblingmolecule

HIH

HIV

VIH

VIV

63

=VIH

VIV

26

=VIH

VIV

H


distributions


Equal

V

H

V

V

H

Observed through emission polarizersSingle rapidly

wobblingmolecule

τ << 4 ns

HIH

HIV

VIH

VIV

12

=VIH

VIV

24

=VIH

VIV


Expressions for Data Analysis

General Expression

Slow Wobble τf << τc << τg

Fast Wobble τc << τf

a = e^ ^

δ

X

Y

θ

φ

Z

s1xs1yp1xp1ys2xs2yp2xp2y

1

2) Verification of method• Single and multi-molecule

polarization of labeled actin• Single and multi-molecule

polarization of RLC-labeledmyosin II

polarized intensities

lab frame

Data Analysis

1) Maximum likelihood fitting routine to convertmeasured intensities to 3-D orientation: θ,φ,δConsiderations include:

• colinear absorption and emission dipoles• evanescent wave polarization • high numerical aperture objective lens• fast and slow motion

a = e^ ^

δ

X

Y

θ

φ

Z

δ

X

Y

Z

α

β

s1xs1yp1xp1ys2xs2yp2xp2y

3

1

2) Verification of method

3) Euler angle transformation

polarized intensities

lab frame

actin frame

Data Analysis

1) Maximum likelihood fitting routine to convertmeasured intensities to 3-D orientation: θ,φ,δConsiderations include:

• colinear absorption and emission dipoles• evanescent wave polarization • high numerical aperture objective lens• fast and slow motion

0

10 0

20 0

30 0

0

50 0

1 00 0

0

3 0

6 0

9 0

0 1 2 3 4 50

3 0

6 0

9 0

s2yp2y

s1xp1x

s1yp1y

s2xp2x

black = calc.blue = fitted

red = βgreen = α

blue = δ

Myosin V - 5µM ATP

Pol

ariz

edFl

uore

scen

ce C

ount

sTo

tal

Inte

nist

yA

ngle

s(d

egre

es)

time (s)

a = e^ ^

α

βδ

1

!!!!!!!Myosin V Beta Distribution!!!!!!!

0

5

10

15

20

25

30

35

0 15 30 45 60 75 90

# E

vent

s

Beta

86 molecules443 events

event duration (sec)0.0 0.2 0.4 0.6 0.8 1.0

# ev

ents

0

20

40

60

80

100

120 b) 40 µM ATP

event duration (sec)0.0 0.2 0.4 0.6 0.8 1.0

# ev

ents

0

5

10

15

20

25

30

35

40a) 5 µM ATP

Event Time Histograms

40 µM ATP

5 µM ATP

38.1 ± 3.5107 ± 9354 ± 54036.4 ± 4264 ± 26138 ± 35

Step(nm)

Dwell time(ms)

Velocity(nm/s)

[ATP](µM)

DD DT

K2 = 12 s-1

TD

k1 = 1.0•106 M-1s-1

D

1 1velocity × + = 38nm/rotationk [ATP] k1 2

⎛ ⎞⎜ ⎟⎜ ⎟⎜ ⎟⎜ ⎟⎝ ⎠

Translation / Tilt

# of

Eve

nts

Event Duration (s)

Event Duration (s)

# of

Eve

nts

laser in laser outθ

φ(-X)

Z

Y

quartz coupling element

quartz slideevanescentwave

Evanescent Wave (TIRF) Microscopy

0

10 0

20 0

30 0

0

50 0

1 00 0

0

3 0

6 0

9 0

0 1 2 3 4 50

3 0

6 0

9 0

s2yp2y

s1xp1x

s1yp1y

s2xp2x

black = calc.blue = fitted

red = βgreen = α

blue = δ

Myosin V - 5µM ATPP

olar

ized

Fluo

resc

ence

Cou

nts

Tota

lIn

teni

sty

Ang

les

(deg

rees

)

time (s)

a = e^ ^

α

βδ

1

Joe Forkey, Margot Quinlan, Alex Shaw, John CorrieNature 2003

Diffraction limited spotWidth of λ/2 ≈ 250 nm

0

40

80

120

160

200

240

280

05

1015

2025

510

1520

25P

hoto

nsX DataY axis

center

width

With enough photons (signal to noise) …Center can be determined to ≈ 1 nm.

Center represents (under appropriate conditions) position of dye.

Myosin V Processivity

20,000 nm10x speed

1000 nm

The Movie

Paul Selvin, Taekjip Haand colleagues

University of Illinois

Center of mass

37/2 nmx

74 nm

37-2x

37 nm

Expected step sizeHand-over-hand:

Head = 2 x 37 nm= 74, 0, 74 nmCaM-Dye: 37-2x, 37+2x, …

0 nm

37+2x

Myosin V Labeling on Light Chain: Expected Step Sizes

0 10 20 30 40 50 60 70 80 90 1000

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

60 65 70 75 80 85 900

10

20

30

40 µ = 74.08 nm σ = 5.25 nmR2 = 0.99346 χ2 = 1.6742 Nmol = 31 Nstep = 227

histogram of 74-0 nm steps

step size (nm)

numb

er of

step

s

75.4 nm

82.4 nm

65.1 nm

83.5 nm

69.8 nm64.2 nm

78.8 nm

70.0 nm70.9 nm

79.1 nm

67.4 nm

68.8 nm73.7 nm

70.7 nm

70.1 nm

68.9 nm71.3 nm

72.0 nm

time (sec)

Posit

ion

(nm

)

higher [ATP](400 nM)

[ATP] = 300 nM

Myosin V steps: 74 nm +/- 5nm

50-20 nm steps

0 10 20 30 40 50 60

0

100

200

300

400

500

600

700

800

900 s1= 23.08 nm σ =3.44 nm Nmol=6s2= 51.65 nm σ = 4.11 nm Nstep=92

73.4 nm

73.5 nm

69.2 nm44.8 nm

24.9 nm

74.4 nm

72.2 nm25.6 nm

58.1 nm18.9 nm

45.5 nm19.2 nm

27.2 nm

74.3 nm

58.6 nm18.7 nm

66.6 nm

82.1 nm

72.5 nm

56.6 nm

68.4 nm

73.5 nm54.1 nm

48.8 nm

15.4 nm48.3 nm

23.7 nm

25.8 nm68.2 nm

20 30 40 50 60 70 800

2

4

no. o

f ste

ps

step size (nm)

time (sec)

Posit

ion

(nm

)52-23 nm steps

C. Hot Spot

37+2x37–2x

37+~

Expected Angles

Expected Angles

Expected Angles

nm37-~

nm

Models for Myosin V ProcessivityA. Hand Over Hand B. Inch Worm

37 37nm

Expected Translocation

Expected Translocation

large wobble

small wobble

large wobble

Hand over Hand Model for Myosin V Processivity

37+2x

x Expected Angles

nmExpected

Translocation37–2x

ADP, Pi

ATP

ADP, Pi

ATP

Coworkers and Collaborators on Single Molecule Fluorescence and Mechanics

Universityof

Pennsylvania

Universityof

Pennsylvania

Universityof

Pennsylvania

John E. T. CorrieDavid R. Trentham

Sheyum SyedErdal Toprak

John BeusangTrey Schroeder Mark ArsenaultYujie Sun

Yuhong WangGraham DempseyRama KhudaravalliJenny Ross

Joe ForkeyMargot Quinlan M. Alex ShawJody DantzigStephanie RosenbergYasuharu Takagi

Henry ShumanErika HolzbaurE. Michael OstapBarry S. Cooperman

Mitsuo IkebeKazuko Homma

Paul R. SelvinTaekjip HaAhmet YildizSean A. McKinney

Myosin V Processivity 1

Deterministic Working Stroke

Thermal Motions

Completion of Step

Lifetime of Myo V S1 Attachments

r = r0 exp(- W / kT)

Veigel et al., Nat Cell Biol. 2005

DD DT

K2 = 12 s-1

TD

k1 = 1.0•106 M-1s-1

D

1 µm

The Movie

ErdalToprak

TaekjipHa

AhmetYildiz

xy

zθ

φ

FluorescenceImaging atOneNanometerAccuracy

DefocussedOrientation andPositionalImaging

PaulSelvin

θax

θay

εy

εx

x

y

z z

y

x

A B

ωey

ωez

cos2(θay)sin2(ωey)

sin2(ωez)

θ

cos2(θax)

φ

70 nm pixels; 2 frames/s

β

α

z

xy

SheyumSyed

Myosin Family Tree

• Myosin-VI is a processive motor that takes frequent backward steps.

• The step size of myosin-VI is much larger than expected, based on the length of the putative lever arm.

Myosin VI

MORE Complicated SMFP Experimental Setup:

xyz

Beam 1 Beam 2Ez

Ex

E45R E45LEz

Ey

xyz

Beam 1 Beam 2Ez

Ex

E45R E45LEz

Ey

6 different polarizations for excitation

s1Ix 45L1Ix s2Ix

s1Iy 45R1Iy s2Iy

p1Ix 45L1Ix p2Ix

p1Iy 45R1Iy p2Iy

Harry TreySchroeder

JohnBeausang

x-APD

y-APD

Fluoroescent Emission

laser

Pockel CellPockel Cell

Beam splitter

sample

-APD

Y-APD

Green Laser

Fluorescent Emission

laser


Beam splitter

sample

x APD

y -APD

Green Laser


2 different polarizations for detection

FlipThreshold 60:

_x_ delta

0 25 50 75 100 125 150 175 200 225 2500

45

90

cycle

_ alpha- Flipped alpha- neg+180

0 25 50 75 100 125 150 175 200 225 250

200

100

0

100

cycle

90

90−

-o- beta

0

45

90

__ s2Iy__ p2Iy

__ s2Ix__ p2Ix

__ 45RIy__ 45LIy

__ 45RIx__ 45LIx

__ ITot__ IFit

end 137=χ .8:=filename "1021e"=pt 36=

coun

ts p

er g

ate

/ ang

le

pnum 19=

βα

δsz

xy

δs

β

α

ITOTIFIT

45RIx45LIx

45RIy45LIy

s2Ixp2Ix

s2Iyp2Iy

Myosin VI 150 µM ATP

Time (s)

FlipThreshold 60:

_x_ delta

0 25 50 75 100 125 150 175 200 225 2500

45

90

cycle

_ alpha- Flipped alpha- neg+180

0 25 50 75 100 125 150 175 200 225 250

100

0

100

cycle

90

90−

-o- beta

0

45

90

__ s2Iy__ p2Iy

__ s2Ix__ p2Ix

__ 45RIy__ 45LIy

__ 45RIx__ 45LIx

__ ITot__ IFit

end 74=χ .8:=filename "1021e"=pt 35=

coun

ts p

er g

ate

/ ang

le

pnum 18=

βα

δsz

xy

δs

β

α

ITOTIFIT

45RIx45LIx

45RIy45LIy

s2Ixp2Ix

s2Iyp2Iy

Myosin VI 150 µM ATP

Time (s)

Molecular Motor Toolbox

Dynein and Dynactin Complex for Cargo Binding

Single Molecule Symposium

April 21, 2005

Rhodamine Microtubules in epi-fluorescence

Processive GFP-dynactin/dynein visualized by total internal reflection fluorescence (TIRF) microscopy.

∆t = 100 ms

Single Molecules of GFP-Dynactin/Dynein Walk Along Microtubules Bi-directionally

Region of Interest 3µm

Analysis using Kymographs of Single Microtubules

Angle, θ, relates to velocity in pixels/frame by: tan(θ) =∆x(pixels)∆t( frames)

3µmD

ista

nce

(pix

els)

Time (pixels)

θ

Conclusion: Dynein Bi-Directionality Caused by Flexible Structure

First direct observation of dynein motility via fluorescence

Dynein AAA motor head could function as a gear

Dynein appear bi-directional: How?

Over-rotation of coiled-coil flexible linkers?

Continues to powerstroke

Walks in opposite direction

Medalia et al. 2002 Science. 298:1209-13.

Artist’s ImaginationCryo-EM of Cell

David C. Goodsell

Universityof

Pennsylvania

Universityof

Pennsylvania

Universityof

Pennsylvania

Vesicle Movement in Cell ExtractNira Pollack & Ron D. Vale, UCSFFrom: Molecular Biology of the Cell, 4th ed.

Melanosome MovementJohn A. Hammer, III, NIH

Molecular Motors in Non-Muscle Cells

Universityof

Pennsylvania

Universityof

Pennsylvania

Universityof

Pennsylvania

Challenges and Opportunities

• Improvement of experimental technology

• Functional modulation the rate constants

• Controlled assembly of secondary structure?

• Mechanisms of the other motors

• Specificity of cargo binding

• Targeting of cargo destination

• Integration with other events (cell division, signal transduction, etc)

Myosin’s Thermal Search

Joe ForkeyMargot Quinlan Stephanie RosenbergJohn BeausangHarry (Trey) Schroeder

Acknowledgements

Universityof

Pennsylvania

Universityof

Pennsylvania

Universityof

Pennsylvania

• Molecular Motors

• Muscle Energetics and Strain Dependence

• Unconventional Myosins – Myosin V

• Single-Molecule Fluorescence Polarization

•

•

• Challenges

APS Workshop on Biology March 12, 2006

[email protected]

SMFP Experimental Setup:

laser


Beam splitter

sample

X-APD

Y-APD

Green Laser


laser


Beam splitter

sample

X-APD

Y-APD

Green Laser


Use polarized light for excitation and detection to determine orientation of probe

xyz

Beam 1 Beam 2Ez

Ex

E45R E45LEz

Ey

xyz

Beam 1 Beam 2Ez

Ex

E45R E45LEz

Ey

6 different polarizations for excitation

2 different polarizations for detection

Totally eight measured intensities along x and y direction obtained within 40 ms

s1Ix (45L1Ix) s2Ix

s1Iy (45R1Iy) s2Iy

p1Ix (45L1Ix) p2Ix

p1Iy (45R1Iy) p2Iy

The image below is a panoramic view of the interior of a eukaryotic cell, such as a cell from your own body. The area covered is shown in the schematic map to the right. The panorama starts at the cell surface, passes through an area of cytoplasm, then follows the synthesis of proteins from the endoplasmic reticulum, through the Golgi, and into a coated vesicle. At the center of the panorama is a mitochondrion, generating energy for the cell. The final region passes into nucleus. All macromolecules are shown, with proteins in blue, DNA and RNA in red and orange, lipids in yellow, and carbohydrates in green. Ribosomes, composed of RNA and protein, are colored magenta. In a real cell, the spaces between each macromolecule are filled with small molecules, ions and water.

Roger Goldman, PGFR USA, 1984

Roger Goldman, Proc. Gold. Fam. Refrig., 1984

Myosin V Strolling

Joe ForkeyMargot Quinlan Stephanie RosenbergHenry ShumanBarry S. Cooperman

Acknowledgements

x

∆G

k+ k-µ2

µ2(x)( ) B2 1[ ]// x k Tk k K e µ µ

− +−= =

B1 2[ ( ) ]/x k TK e µ µ−=

1 0( ) ( )x F x dxµ µ= +∫

Rate and Equilibrium ConstantsDepend on Mechanical Strain

University APS Workshop on Biology of Pennsylvania March ... · APS Workshop on Biology March 12,...

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