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)

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)

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

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

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

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.

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

Surface functionalized

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

blocking agent

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

F-actin Linker to F-actin

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. 47. 33179–33182, (1999)

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, 8635-8638 (2006)

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

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

Photo pattern reagent

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)

Homogenous linker

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), 269-277

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

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

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

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

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

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

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

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. )

Streptavidin Flexible structure Flexible structure )

F-actin caha )

Function of motor protein 1 Actin filament and Protein motors (Myosin) Function of motor protein