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Force measurement:Molecular motor
&Cancer cell migration
Takeshi SakamotoAssociate ProfessorDepartment of Physics and AstronomyWayne State University
Takeshi SakamotoAssociate ProfessorDepartment of Physics and AstronomyWayne State University
Force measurement:Molecular motor
&Cancer cell migration
My labs focus on..
(1) Determine the force-generation map of cancer cell; controlling metastasis
(2) Determine the mechanical property of the motor by using TIRF microscope and optical trap
My labs focus on..
(1) Determine the force-generation map of cancer cell; controlling metastasis
(2) Determine the mechanical property of the motor by using TIRF microscope and optical trap
Metastasis of the cancer cell&
New protein, TRIOBP, in cancer
Traction Force Microscope
My labs focus on..
(1) Determine the force-generation map of cancer cell; controlling metastasis
(2) Determine the mechanical property of the motor by using TIRF microscope and optical trap
Single molecule motility assay
Single molecule Force measurement by optical tweezers
My labs focus on..
(1) Determine the force-generation map of cancer cell; controlling metastasis
(2) Determine the mechanical property of the motor by using TIRF microscope and optical trap
Single molecule motility assay
Single molecule Force measurement by optical tweezers
The function of myosins are very wide variety
Muscle contraction
The size of things
What kind of motor in our body?
Myosin
Kinesin
Dynein
Rotation motor
Yasuda R, Cell, 1998
Conventional myosin
Myosin V: a cargo transportermelanosome, mRNA,
endoplasmic reticulum
Cytokinesis
Vesicle transportation
Humans have
39 myosins
from 12 classes
From Berg et al. Mol. Biol. Cell 12: 780, 2001
Myosin Motor Family in Humans
Myosin V
○ N-terminus motor domain,○ 6 light chain binding sites
(CaM or light chain (ELC))IQxxxRGxxxR : 23~25 aa
○ Tails have Coiled-coil andglobular domain
○ Does not form filaments
♠ Transport cargo♠ Processive motor
Walker M., et al., 2000, Nature, 405
Myosin V
Actin filament
○ N-terminus motor domain,○ 6 light chain binding sites
(CaM or light chain (ELC))IQxxxRGxxxR : 23~25 aa
○ Tails have Coiled-coil andglobular domain
○ Does not form filaments
♠ Transport cargo♠ Processive motor
Myosin V
Actin filament
Motor domain
Actin bingin domainATP bindingATP hydrolysis energy convert into force
Myosin V
Actin filament
Neck region and light chain
-helix on heavy chain6 IQ motifs: IQxxxRGxxxR,
23~25 aminoacidLight chain: Calmodulin
Essential light chainMeasurering stiffness
Lever arm
Myosin V
Actin filament
Neck region and light chain
Coiled-coil : dimerize two heavy chain
Myosin V
Actin filament
Neck region and light chain
Coiled-coil
Globular tail :cellular functionsBinding another protein: Rab27a
Courtesy ofJohn Hammer andXufeng Wu, NIH
The “Dilute” Mouse is a Good Model for Griscelli’s Disease
Wild type Dilute Melanocytes
Courtesy ofJohn Hammer andXufeng Wu, NIH
The “Dilute” Mouse is a Good Model for Griscelli’s Disease
Wild type Dilute Melanocytes
Wu X and Sakamoto T, et al. , FEBS Lett.
580 :5863-8, 2006.
Movement of the myosin V-melanophilin-Rab27acomplex
Cy3-CaM GFP-Rab27a
4 m
Rhodamine-actin with GFP-Rab27a
GFP-Rab27a
Total Internal Reflection Fluorescent Microscopy (TIRF) and
single molecule motility assay
Ez = Eo exp(-z/dz)
2n
dz =
Penetration depth of evanescent wave
Wide-field Fluorescence TIRF
Advantage of TIRF Microscopy
Total Internal Reflection Fluorescent Microscopy
10x real time
Fluorescent Imaging at One Nanometer Accuracy
FIONA
Yildiz A, et. al., Science, 2003
>40,000 photons ~ 1.5 nm accuracy
510152025303540
-50
0
50
100
150
200
5 10 15 20 25 30 35 40
Conditions: < 300 nM ATP
Recently, Dr. Warshaw have demonstrated that heads ofmyosin V alternatively steps on actin filament.
Stepping Trace of Myosin V longer neck (8IQ) mutant
300nM ATP
6 IQ
Three models of myosin V movement
Tight couplingCenter of mass 36nmEach head steps 72nm
Diffuse movement ~36nm
Current 5.5 nm x 3 = 16.5 nmOkada T et al., 2007, B.B.R.C
step size 36nm
Tractor beam in the space
Trapping beam in the lab
Trap an object by LASER light
LASER trapping for 1 µm spherical bead
Monitor
Optical trap study of single myosin VIn vitro motility study
Mehta AD, Nature. 1999;400:590-3
My labs focus on..
(1) Determine the force-generation map of cancer cell; controlling metastasis
(2) Determine the mechanical property of the motor by using TIRF microscope and optical trap
Single molecule motility assay
Single molecule Force measurement by optical tweezers
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