"Experiments & Results"

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Marimo gel

Preparation of spinach thylakoid membranes

All purification procedures were performed at 4 ^oC. 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.)

Preparation of Marimo-gel by N₂ blow

Experiment

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.


File:Making marimogel

(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.)

Results

From the photograph and histogram, it is evident that Marimo-GEL of large size (>300 µm diameter) could be prepared,
which not small enough by introducing into the flow cell.

Preparation of Marimo-gel by using micro emulsion

Experiment

0.2 g/mL ER290 surfactant in paraffin oil 1000 µL was added on the mixture of 2 wt % alginate 100 µL and
2.1 mg/mL thylakoid membrane 100 µL in the brown bottle. The brown bottle was vortexed to form micell which was composed of
the mixture of alginate and thylakoid membrane. The micell in the dispersion liquid was added on the 50 mM BaCl₂ solution in a
microcentrifuge tube. When the tube was centrifuged, Marimo-GEL was formed due to cross-link between alginate and Ba²⁺ ions.

Results

From the photograph and histogram, it is evident that Marimo-GEL of very small size (<100 µm diameter) could be prepared,
which small enough by introducing into the flow cell.

Synthesis of ATP

Preparation of ATP standard curve

Experiment

1 mg of Luciferase (Sigma Aldrich) was dissolved in 10 mL of the HEPES buffer containing 15 mM MgSO₄ at pH 7.8. The pH
was adjusted using NaOH. 1 mg of luciferin was dissolved in 500 µL of the HEPES buffer. 20 µL of ATP at 5, 20, 50, 100, 200 mM
was mixed with 20 µL of luciferin solution, 10 µL of luciferase solution, and 150 µL of the HEPES buffer. Photon production
was measured by using a photon counter (Scientex) after 1 minute from mixing the ATP and luciferin-luciferase. The amount of different
ATP concentration was determined. Standard solutions were made by using ATP(Wako).

Determination of the ATP concentration

Experiment

Marimo gel suspension was placed on the coverglass, and 455-485 nm light was irradiated for 4.5 h. The supernatant was mixed with
luciferin-luciferase solution. The amount of light of any given onentration of ATP was determined.

Movement of microtubule

Preparation of ATP standard curve

Experiment

Flow-cells were prepared by placing a coverglass (18 × 18 mm²) on a coverglass (24 × 60 mm²) equipped
with a pair of double-sided tape to form a chamber of approximately 2 × 18 × 0.1 mm³ (W × L × H) in dimension.
The flow cell was filled with casein solution (80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, 0.5 mg mL^-1 casein; pH adjusted to 6.8
using HCl). After incubating for 2 min with casein to mask the glass surface, 5 µL of 200 nM kinesin solution (80 mM PIPES,
1 mM EGTA, 1 mM MgCl₂, 0.5 mg mL^-1 casein, 1 mM DTT, 10 mM paclitaxel, 1% DMSO; pH 6.8) were introduced
and incubated for 5 min to bind the kinesins to the casein coated surface. The flow cell was washed with 5 µL of motility buffer.
5 µL of MTs solution (250 nM in motility buffer) was then introduced and incubated for 2 min, followed by washing with 5 µL of
motility buffer. Finally, the gliding motion of microtubules was initiated by applying 5 µL of the　different concentration of
ATP buffer(0.0005, 0.001, 0.0025, 0.005, 0.05, 0.5 mM ATP) and measured velocity of microtubule. This experiment was performed at room temperature.

Results

We prepared standard curve to calculate velocity of microtubules with ATP concentrations. We observed a linear relation between
ATP concentration and microtubule velocity at low ATP concentration. This linear relation will be used for determining the concentration
of ATP synthesized from Marimo-GEL by monitoring the velocity of microtubules.



Dependence of velocity of microtubules on the concentration of ATP (left) and initial part of the graph (right).

in vitro Motility assay using the ATP produced by Marimo gel

Experiment

BaCl₂ solution in which Marimo-gels were soaked was substituted with motility buffer. 50 µL of Marimo-gels dispersion was
mounted on the cover glass, and was exposed to 455-485 nm light for 4.5 h. 0.5 µL of 450 mg mL^-1 D-glucose, 0.5 µL of 5000 U mL^-1
glucose oxidase, and 0.5 µL of 5000 U mL^-1 catalase were added to Marimo-gels dispersion. And then, this solution was centrifuged.
Flow-cells were prepared by placing a coverglass (18 × 18 mm²) on a coverglass (24 × 60 mm²) equipped with a pair of
double-sided tape to form a chamber of approximately 2 × 18 × 0.1 mm³ (W × L × H) in dimension. The flow cell was filled with casein solution
(80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, 0.5 mg mL^-1 casein; pH adjusted to 6.8 using HCl). After incubating for 2 min with casein to mask
the glass surface, 5 µL of 200 nM kinesin solution (80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, 0.5 mg mL^-1 casein, 1 mM DTT, 10 mM paclitaxel,
1% DMSO; pH 6.8) were introduced and incubated for 5 min to bind the kinesins to the casein coated surface. The flow cell was washed
with 5 µL of motility buffer. 5 µL of MTs solution (250 nM in motility buffer) was then introduced and incubated for 2 min, followed by
washing with 5 µL of motility buffer. Finally, the gliding motion of microtubules was initiated by applying 5 µL of the supernatant after
centrifugation of Marimo-gels solution. This experiment was performed at room temperature.

Results

We observed movement of microtubules and measured velocity of microtubule. Average velocity of microtubules was 0.06 µm/s and we estimated
ATP concentration was 0.0065 mM by using ATP standard curve.


File:GFP microtubule
Average velocity = 0.06 µm/sec
ATP conentration = 0.0065 mM
Fluorescent image of microtubule driven by kinesin utilizing the ATP regenerated by Marimo gel.

Mari-motor system

Experiments

First, BaCl₂ solution in which Marimo-gels were soaked was substituted with motility buffer. Next, flow-cells were
prepared by placing a cover glass (18 × 18 mm²) on a cover glass (24 × 60 mm²) equipped with a pair of double-sided tape
to form a chamber of approximately 2 × 18 × 0.1 mm³ (W × L × H) in dimension. The flow cell was filled with casein solution (80 mM PIPES, 1 mM EGTA,
1 mM MgCl₂, 0.5 mg mL^-1 casein; pH adjusted to 6.8 using HCl). After incubating for 2 min with casein to mask
the glass surface, 5 µL of 200 nM kinesin solution (80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, 0.5 mg mL^-1 casein, 1 mM DTT, 10 mM paclitaxel,
1% DMSO; pH 6.8) were introduced and incubated for 5 min to bind the kinesins to the casein coated surface. The flow cell
was washed with 5 µL of motility buffer. 5 µL of MTs solution (250 nM in motility buffer) was then introduced and incubated
for 2 min, followed by washing with 5 µL of motility buffer. Finally, 5 µL of the Mario-gels dispersion (include 100 nM phenazine
methosulfate) was applied. This experiment was performed at room temperature. For observing microtubules, samples were illuminated
by green light (528-552 nm) with a 100W mercury lamp and visualized by epifluorescence microscopy using an oil-coupled Plan
Apo 60×1.40 objective, where exposure time was maintained of 300 ms. Also for synthesizing ATP, Marimo-Gel was illuminated
by blue light (455-485 nm), and exposure time was 5 s. We went time-lapse observation interval of 10 seconds.