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