"Materials"
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.
Preparation of brain tubulin
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.)
Machines
Microscope
To study the motility of MTs, we used a 100 W mercury lamp for illuminating of samples and
an epifluorescence microscope (Eclipse Ti, Nikon) using an oil-coupled Plan Apo 60× objective (Nikon)
for visualizing of samples. Also we used UV cut-off filter blocks (GFP-HQ: EX455-485, DM495, BA500-545; Nikon)
in the optical path of the microscope; these blocks allowed visualization of samples but eliminated the UV portion of the radiation, thus
minimizing the harmful effect of UV radiation on the samples.
Moreover we connected a cooled-CMOS camera (NEO sCMOS, Andor) to a PC for capturing images.
Optical tweezer
Optical tweezer is a scientific instruments which can hold and move microscopic objects
by using a highly focused laser beam. It provides an attractive or repulsive force (typically on the order of pN),
depending on the refractive index mismatch. In this work, we will measure the force of
micro gear by using an optical tweezer.
Here we use a Nd:YAG laser (1064 nm wavelength) to trap a biological specimens.
This is because such specimens (being mostly water) have a low absorption coefficient at this wavelength.
A low absorption is desirable so as to minimize damage of the biological specimens,
which sometimes referred to as opticution. Moreover He:Ne Pilot laser(633 nm wavelength)
was used with Nd:YAG laser for visualization of specimens.
Capturing images were sent to a PC and edited by imaging software (Nikon NIS Elements).
