"Materials"

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MT ring

Preparation of biotin labeled tubulin

1. Tubulin was thawed, and polymerized at 37 °C for 15 min.
2. Biotin-XX-SE (Molecular Probes, Cat. B-1606) was dissolved at 0.1 M in dry dimethyl sulfoxide (DMSO).
3. Biotin-XX-SE solution was added to tubulin, while pipetting to distribute it rapidly to a final concentration of 2 mM. incubate at 37 °C for 20 min.
4. Mixture was layered onto cushions and spun at 54,000 rpm for 1h at 37 °C.
5. Pellets were resuspended and spun cold, being careful to wash the cushion inter face well to remove all the biotin-XX-SE.
6. Tubulin was depolymerized and spun 40,000 rpm for 15 min at 4 °C.
7. Steps (3) – (6) were performed to give once cycled biotin-tubulin.
8. Steps (3) – (6) were repeated to give twice cycled biotin-tubulin. The final pellet is resuspended in BRB80.
9. The final biotin-tubulin is frozen and stored as per the cycled tubulin.

Determination of stoichiometry
Tubulin concentration was determined by SDS-PAGE electrophores.
To quantify biotin, we use defference of avidin and avidin-biotin complex in
spectroscopic characteristic because avidin combines stoichiometrically with biotin.
The dye 4-hydroxyazobenzene-2’-carboxylic acid (HABA), which binds only to avidin with changing spectrum,
so that it can be used as an indicator for unoccupied binding sites [1].

1. Prepare standard curve.
Biotin standard solutions [(0, 10, 20, 50, 100, 200, 300, 500 µM) biotin,
80 mM PIPES, 5 mM MgCl₂, 1 mM EGTA] were prepared.
Each biotin standard solutions was added to avidin solution of which final concentration is
0.4 mg mL-1 avidin, 250 mM HABA, 80 mM PIPES, 5 mM MgCl₂, 1 mM EGTA.
The mixtures were left for 15 min at roomtemperature.
A500 of these solutions were measured to prepare standard curve.

2. Determination of biotin.
1　mg mL-1 Pronase was added to biotin-labeled tubulin, and left for 1 h at 37 °C.
This mixture was added to avidin solution, and left for 15 min at roomtemperature.
A500 of this solution were measured to determine concentration of biotin.

Mechanism of constructing ring shaped microtubules

Surface-adhered kinesin motor proteins can transport biotinylated microtubules.
With adding streptavidin (St), partially St coated microtubules were collide
and collisions of the moving microtubules were enables to cross-linking
when gliding microtubules were encountered.

biotin-streptavidin

Biotin, a 244 dalton compound, is bound with exceptionally high affinity (Ka = 2.5x10¹³ M)
by the 53 kDa protein Streptavidin, due to the extremely slow unbinding rate of the bond.

MARIMO gel

Preparation of spinach thylakoid membranes

All purification procedures were performed at 4 °C.
Thylakoid membranes were prepared from spinach leaves by the modified method of Yu et al.
The spinach leaves ( ~160 g) were washed with deionized water and homogenized in 500 mL of homogenization solution
(0.3 M sucrose, 20 mM NaCl, 5 mM MgCl₂, 50 mM Tris-HCl, pH was adjusted to 7.6)
for 40 sec using an AM-10 homogenizer (Nihon Seiki Seisakusho, Japan).
The homogenate was filtered through four-time folded gauze.
The flow through was suspended in 400 mL of a high ionic strength buffer (10 mM Hepes-KOH, 150 mM NaCl, pH8).
The suspension was centrifuged at 10,000g for 20 min and the precipitate was resuspended in 15 mL
of homogenization solution including 5% DMSO and flash frozen in liquid nitrogen, and stored in liquid nitrogen.

(Yu A. H. C; Hosono K. Biotechnol. Lett. 1991, 13, 411.)

Entrapment of thylakoid membranes in alginate beads:

This procedure is based on the method described by Paul F. et al. and Zekorn T. et al.
A 0.3 mL of suspension of thylakoid membrane containing 1.41 mg protein/mL and 2.7 mL of
a 2%(w/v) sodium alginate solution (100 mM K3PO4, 100 mM MgCl2, 100 mM NaCl, 500 mM PIPES,
200 mM ADP, pH was adjusted to 7.6) were placed in a 1 mL syringe. Using syringe pump (HA2000P, Harvard apparatus, US),
the alginate solution including thylakoid membrane was slowly ejected from the needle and was blown by a nitrogen gas.
The tear shaped green droplet firstly encountered mineral oil phase and transformed into globular shape.
Then the droplet sunk into the second phase, which contains 50 mM BaCl₂ and cross-linkage of
alginate with barium occurred. Because leak was not observed even after few weeks from the encapsulation,
the cross-linked alginate mesh seemed to be enough small to support thylakoid membranes.

(Paul F.; Vignais P. M. Enzyme Mcrob. Technol. 1980, 2, 281.)
(Zekron T.; Horcher A.; Siebers U.; Schnettler R.; Klock G.; Hering B.; Zimmermann U.;
Bretzel R. G.; Federlin K. Acta Diabetol, 1992, 29, 99.)