Experiment 1

Aim
　To investigate the effect of depletion force caused by methylcellulose on the behavior of microtubules

Materials
・Anti-GFP antibody (0.2 mg/mL)
・Motility buffer (80 mM PIPES, 1 mM EGTA, 2 mM MgCl2, 0.5 mg mL−1 casein, 1 mM DTT, 10 μM paclitaxel and ∼1% DMSO; pH 6.8)
・GFP-tagged kinesin solution (consisting of the first 560 amino acids of human kinesin1,80 mM PIPES, 1 mM EGTA, 2 mM MgCl2, 0.5 mg/mL casein, 1 mM DTT, 10 μM paclitaxel/DMSO, ∼1% DMSO; pH 6.8)
・Rhodamine-labelled tubulin solution (0.2 µM tubulin and 1 mM GTP.)
・ATP buffer (∼80 mM PIPES, 1 mM EGTA, 2 mM MgCl2, 0.5 mg/mL casein, 1 mM DTT, 10 μM paclitaxel/DMSO, ∼1% DMSO; pH 6.8)
・MC (0.1wt% or 0.3 wt% Methylcellulose)
・MAP4 (0.25 μM Microtubule-associated protein 4)

Methods
①Flow cells were assembled from two cover glasses, and a double-sided tape was used as the spacer.
②2 μL of anti-GFP antibody was applied to the flow cell. After incubation for 3 min, the flow cell was washed with 5 μL of motility buffer.
③Then, 3 μL of a GFP-tagged kinesin solution was introduced and incubated for 3 min to bind the kinesin to the antibody.
④The flow cell was washed with 5 μL of motility buffer.
⑤3 μL of microtubule solution of a prescribed concentration was introduced and incubated for 3 min, followed by washing with 5 μL of motility buffer 2 times.
⑥MC, 0.25 μM MAP4 and 10 μL of 10 mM ATP buffer was added to the flow cell.
⑦The samples were visualized with a fluorescence microscope using an oil-coupled Plan Apo 60 × 1.40 objective.

Result
　We observed a depletion force among microtubules by adding different concentrations of MC to a solution of microtubules. In lower density of microtubules, prepared from 200 nM tubulin, were suspended in 0.0, 0.1 and 0.3 wt% MC solution and observed under a fluorescence microscope. At the concentration of 0.1 and 0.3wt% of the MC solution, microtubules aggregated because of the depletion force. On the other hand, microtubules moved separately and showed little interaction each other in the absence of MC (0.0 wt%).

Fig.1 Fluorescence microscopy images of microtubules which were suspended in solution (left) without MC and (center and right) with MC of 0.1 and 0.3 wt%. Scale bar: 10 µm

Discussion
　This result indicates that the depletion force induced by MC causes the attractive interaction among the microtubules. We added 0.3% MC to the solution of microtubules in following experiments of gliding assay because we could observe aggregation the most in this condition.

Experiment 2

　Microtubules move separately and there is little interaction among them when there are few microtubules in a flow cell. We assume that the density of microtubules is a key element for the appearance of collective motion.

Aim
　To see the difference of collective motion whether the density of microtubules is high or low.

Materials
・Anti-GFP antibody
・Motility buffer
・GFP-tagged kinesin solution
・Rhodamine-labelled tubulin solution (Low concentration : 0.2 µM, High concentration : 5.0 µM in the presence of 1 mM GTP.)
・ATP buffer
・MC (0.3 wt% Methylcellulose)
・MAP4

Methods
①Flow cells were assembled from two cover glasses, and a double-sided tape was used as the spacer.
②2 μL of anti-GFP antibody was applied to the flow cell. After incubation for 3 min, the flow cell was washed with 5 μL of motility buffer.
③Then, 3 μL of a GFP-tagged kinesin solution was introduced and incubated for 3 min to bind the kinesin to the antibody.
④The flow cell was washed with 5 μL of motility buffer.
⑤3 μL of microtubule solution of a prescribed concentration was introduced and incubated for 3 min, followed by washing with 5 μL of motility buffer 2 times.
⑥0.3 wt% MC, 0.25 μM MAP4 and 10 μL of 10 mM ATP buffer was added to the flow cell.
⑦The samples were visualized with a fluorescence microscope using an oil-coupled Plan Apo 60 × 1.40 objective.

Result
　In order to find out how a density of microtubules affect their collective behavior, we prepared two types of tubulin solution which concentration were 0.2 and 5.0 µM. In high density, microtubules moved randomly but showed collective motion which made large streams at last. However, coordinated behavior was not observed in low concentration tubulin solution.

Fig.2 Fluorescence microscopy images of microtubules at low (left) and high (right) density. Scale bar: 50 µm

Discussion
　In low density, microtubules move more isotropically, that means they move separately. 　And in high density, we can see nematic phase, which means they move as a group. Compared to the one with low density, now microtubules move more as a group making some roads. We successfully reproduce crowding situation in micro scale

＊Experiment 1&2 were done on a flowcell (2×5×0.15mm3　W×L×H) which consists of double-sided tape and a cover glass.

Experiment 3

Aim
　Design flow cells by KLayout to estimate the optimum placement.

Methods
　We designed 4 groups of pillars which each group consisted with circle, two types of triangle, and quadrangle pillars. This time, we used a software named KLayout for design.
　A size of whole cell was 2000㎛×2000㎛, and a size of each pillar group was 7000×7000㎛, and distances between the groups were 2000㎛.
　The whole image of flow cell is as below;

　Each group consisted with 5 areas which distances among pillars were 100㎛~500㎛. The whole image of each group is as below (e.g. ❶circle);

　In each area, we arranged pillars, which diameters were 10~50㎛, in regular interval as following figures;








　We output the blueprint that designed by Klayout and ordered this flow cell from Nanotechnology Platform in University of Tokyo.

Results(microscopic observation)
　The flow cells were visualized with an optical microscope as following images.

Quadrangle pillars




Triangle pillars




Circle pillars

Experiment 4

Aim
　To confirm whether pillars can control collective motion of microtubules

Materials
・PDMS (Dimethylpolysiloxane as flow cell)
・Anti-GFP antibody
・Motility buffer
・GFP-tagged kinesin solution
・Rhodamine-labelled tubulin solution (5.0 µM in the presence of 1 mM GTP)
・ATP buffer
・MC (0.3 wt% Methylcellulose)
・MAP4

Methods
①2 μL of anti-GFP antibody was applied to the flow cell. After incubation for 3 min, the flow cell was washed with 5 μL of motility buffer.
②Then, 3 μL of a GFP-tagged kinesin solution was introduced and incubated for 3 min to bind the kinesin to the antibody.
③The flow cell was washed with 5 μL of motility buffer.
④3 μL of microtubule solution was introduced and incubated for 3 min, followed by washing with 5 μL of motility buffer 2 times.
⑤0.3 wt% MC, 0.25 μM MAP4 and 10 μL of 10 mM ATP buffer was added to the flow cell.
⑥The samples were visualized with an fluorescence microscope using an oil-coupled Plan Apo 60 × 1.40 objective.

Result
　The results of gliding assay using a flowcell made of PDMS are following images. Collective motion was observed as in experiment 2. Around pillars microtubules showed a motion like avoiding obstacles. It was observed that specific distances of pillars made more clear streams.

Fig.3 Fluorescence microscopy images of microtubules in PDMS flowcell.

Discussion
　We succeeded in reproduction of the flow of a human crowd on a flowcell. It became clear that whether a stream close to human crowd appears depends on a distance between each pillar. From the result of this experiment, pillars at a distance of about 40㎛ are thought to be appropriate to reproduce a clear stream.