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
(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
PennPennPennForce-Velocity, Power and Energy Utilization
W●
H●
W ●
+
W = Power = F dx/dt = F · V●
Fenn and Hill, J. Physiol. 1932
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
Power Relative to P Vo max
PennPennPenn
W●
H●
W ●
+
W = Power = F dx/dt = F · V●
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
PennPennPennForce-Velocity, Power and Energy Utilization
W●
H●
W ●
+
W = Power = F dx/dt = F · V●
Fenn and Hill, J. Physiol. 1932
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
Photoselectedorientation
distributions
Observed through emission polarizers
Overallorientationdistribution
HIV
V
H
V
V
H
Excitation Detection
HIV
VIH
VIV
H
Photoselectedorientation
distributions
Observed through emission polarizersOverall
orientationdistribution
HIH
V
H
V
V
H
Excitation Detection
HIV
VIH
VIV
H
Photoselectedorientation
distributions
Observed through emission polarizersOverall
orientationdistribution
HIH
V
H
V
V
H
Excitation Detection
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
Emission polarization ratios
Equal
HIH
HIV
VIH
VIV
13
=VIH
VIV
26
=VIH
VIV
Excitation Detection
H
Orientation distributions of
several excitations
Observed through emission polarizers
4 ns << τ << 10 ms V
H
Excitation
V
V
H
Detection
Emission polarization ratios
not Equal
Single slowly wobblingmolecule
HIH
HIV
VIH
VIV
63
=VIH
VIV
26
=VIH
VIV
H
Photoselectedorientation
distributions
Emission polarization ratios
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
Excitation Detection
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
Myosin V Processivity 2
Myosin V Processivity 3
Myosin V Processivity 4
Deterministic Working Stroke
Thermal Motions
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
Pockel CellPockel Cell
Beam splitter
sample
x APD
y -APD
Green Laser
Fluorescent Emission
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
SMFP Experimental Setup:
laser
Pockel CellPockel Cell
Beam splitter
sample
X-APD
Y-APD
Green Laser
Fluorescent Emission
laser
Pockel CellPockel Cell
Beam splitter
sample
X-APD
Y-APD
Green Laser
Fluorescent Emission
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
Top Related