2. What is F-actin ? ・Unidirectional structure ・Composed of G-actin
(actin subunit)
Polymerization
(-)Low growth
(+)Fast growth
F-actin
(actin filament)
・Unidirectional structure
・Composed of G-actin
Molecular Biology of the Cell (© Garland Science 2008)

3. Example of “transport” by protein motors (myosin-V) in vivo
Actin filament and Protein motors (Myosin)
Example of “transport” by protein motors (myosin-V) in vivo
mRNA
localized
Linker protein
In vitro, protein motors exhibit same function as beading assay.
Molecular Biology of the Cell (© Garland Science 2008)

4. protein motors for Nano device
Beading assay
Motor protein walk on filament
cargo
myosin
F-actin
In vitro
The future innovation
・Use some type of motor protein
In vitro , transport intelligent machine
・Use natural cargo link
・Control by other kind protein

5. Problem of F-actin control on surface
Previous myosin beading assay
myosinV beading assay
On Paracrystal F-actin
AFM image of paracrystal F-actin
F-actin on surface show random assembly
In vitro assay

6. protein motors for Nano device
Beading assay
Motor protein walk on filament
cargo
myosin
F-actin
In vitro
Goal of this group
Create a unidirectional F-actin track
for the long-distant movement of Myosin-bead

7. protein motors as Nano device
a unidirectional F-actin track for the long-distant movement of Myosin-bead
• To make long ”roads” for myosin
-control length of each F-actin
-surface blocking reagent selection
-photo-patterned of F-actin
-fluidic flow or electric field*
* Nanotechnol. Mater. Dev. Conf. (2009)245.

8. F-actin
Photo patterned agent
Linker to F-actin
Homogenous linker
Blocking agent

9. Surface functionalized

10. photo-patterned glass surface
NH2 group functionalized
APTES
After APTES treatment
Before APTES treatment

11. blocking agent

12. on protein linker surface
photo-patterned glass surface
blocking agent
Block effect
on ATPES surface
Block effect
on protein linker surface

13. F-actin
Linker to F-actin

14. To pattern F-actin on glass
Gelsolin
Gelsolin
F-actin
• sever F-actin
•after severing , Gelsolin cap F-actin
•Capping state is stable. Ka=109 (M-1)
• in solution, Gelsolin concentration
control the length of F-actin
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No –33182, (1999)

15. To pattern F-actin on glass
Gelsolin for F-actin linker
1,Gelsolin was functionalized　 by “anchor” unit ,biotin
2, mixture with F-actin and　biotinylated Gelsolin
Langmuir 22, (2006)

16. Control F-actin length by gelsolin
Biotinyled-Gelsolin concentration (Cg) change image
Molar Ratio (F-actin:gelsolin)
1:400
1:40
1:4
F-actin concentration is constant
To create strict surface Randomly
Glass surface is coated by + charged polymer ,PDDA

17. Control F-actin length by gelsolin
Molar Ratio (F-actin:gelsolin)
(3times observation)
Control length gradually

18. Photo pattern
reagent

19. photo-patterned glass surface
Photo-patterned by photo-biotin
Photo-biotin
azido (N3) reaction with NH2 group by light (λ=365nm)
Photo mask (Line features -60µm thick)
Biotin unit link to streptavidin (homogenous linker)

20. Homogenous linker

21. photo-patterned glass surface
Streptavidin
Biotin binding
Streptavidin
Streptavidin
•4 binding-sites to biotin
•Strong non-covalent bond with biotin Ka=1015(M-1)
• fluorescent dyed labeled
Trends in Biotechnology Vol25, Iss6, (2007),

22. photo-patterned glass surface
Photo-patterned glass image
By fluorescents of streptavidin

23. F-actin
Photo patterned agent
Linker to F-actin
Homogenous linker
Blocking agent

24. patterned F-actin on glass
Fluorescent microscope image
The number of F-actin
On different area
F-actin
Image
(Rhodamine)
(λ ex=580(nm))
Streptavidin
Image
(FITC)
(λ ex=521(nm))
White bar- direct counting
Black bar- wash out 1time

25. Control of transport as rail
Conclusion and Future research
• To make long ”roads” for myosin
-control length of each F-actin
-F-actin length average 2.5um
-surface blocking reagent selection
-BSA show blocking effect
-photo-patterned of F-actin
Curve shape transport
Control of transport as rail

26. actin structure )
G-actin
(monomer state)
F-actin
(polymer state)

27. APTES modification After APTES coat Clean up glass
Acetone in ultrasound bath
Methanol in ultrasound bath
Dried with N2 gas
APTES coat
In ultra sonic bath (10min)
Before APTES coat
APTES in anhydrous ethanol(3% v/v)
Incubated for 12 hours
Oven for 10min ,at 125ºC

28. F-actin (polymer state) Flexible structure
A long-distant directed F-actin array
e.g2500um
Flexible structure )
F-actin
(polymer state)

30. Streptavidin (A) nonbiotinylated F-actin bound to a bare gold surface.
(B) biotinylated F-actin bound to a streptavidin-modified gold surface. Each bright dot the labeled actin fixed onto the streptavidin surface. Some of the dots are marked by dotted circles.
(C) LFM image of ODT (dark regions) and cysteamine (bright regions) SAM patterns on a gold surface (no actin).
(D) Snapshot of a movie showing biotinylated F-actin assembled onto streptavidin patterns (two bright stripes). ODT patterns (dark stripes) blocked the nonspecific binding of biotinylated F-actin. Some dots representing fixed actins are marked by dotted circles. )

31. Streptavidin
Flexible structure
Flexible structure )

32. F-actin caha )

33. Function of motor protein1
Actin filament and Protein motors (Myosin)
Function of motor protein