User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/06/01/A Polarized Microtubule Array for Kinesin Powered Nanoscale Assembly and Force Generation

Disclaimer

This is my interpretation of the following article. Please do not accept my comments as correct until you have read the paper and can generate your own thoughts on it.

Paper

A Polarized Microtubule Array for Kinesin-Powered Nanoscale Assembly and Force Generation

Review

Purpose

• They are trying to make aligned microtubules. The reason they want to do this is to try and move the idea of using microtubules and kinesin in microscale devices from science fiction, to reality.

Notes

• They give the size of a conventional kinesin (I think it's called kinesin-1) 7nm x 4 nm x 4nm Reference
• They note that patterning the substrate microtubules are on will cause them to move in only one direction. Reference
  • Note: We could definitely do this at CHTM. It could also change kinesin processivity.
• They briefly describe the best way to pattern a substrate for microtubule directed movement. It includes:
  • Lithographically pattern arrowhead shapes 1 µm thick in photoresist on glass.
• They also mention 2 other groups that have tried to direct microtubule assembly. One group forced the microtubules to align in one direction by flowing fluid in a flow cell in a gliding motility assay. Another group fixed the minus end of the microtubules to a coverslip and then flowed fluid in a chamber to align them in one direction. The only problem was that they couldn't fix the microtubules after flow started.
• Their idea to align microtubules was to grow a fixed seed and then polymerize microtubules from that seed.
• Method
  • They used BRB80 as their buffer.
  • They used Taxol as the fixing agent.
  • They polymerized using standard procedures.
  • They found that APTES gave the highest density of surface immobilized microtubules.
  • Another technique used to immobilized microtubules was to use kinesin bound to glass and then use the ATP analog AMP-PNP.
  • They used casein to passivate surfaces. It's funny but they show that casein actually prevents microtubules from growing where the casein is.

• They grew microtubules and then flowed them in a flow cell to align them and then they added kinesin with AMP-PNP to bind the microtubules to the glass. (Very cool).
• They also used 1% gluteraldehyde to fix the microtubules even more I guess.

• They found that the critical concentration of tubulin for nucleating microtubules was 10 µM while for growth from existing microtubules was only 1 µM.
• So cool, there exists a modified tubulin complex called NEM tubulin that prevents minus end polymerization while doing nothing to the positive end polymerization of microtubules. Reference
• Results
  • They did it! Using their technique of:

1. Preparing a casein coated coverslip with a segment of it coated with tape to prevent casein from attaching there.
2. Using APTES to coat the uncoated casein segment.
3. Seeding microtubule growth.
4. Growing microtubules.
5. Flowing in a chamber that causes the free end of the microtubules to bend towards the coverslip.
6. Flowing in kinesin with AMP-PNP to bind to the free end of the microtubules and correspondingly to the casein coated region of the coverslip.

• They were able to get 9 µm segments of microtubules to stay straight with about 12˚ of deviation.
• They note that this technique is not good enough to do molecular machine stuff.

Take home

Very nice article that references other techniques to make patterned microtubule arrays. I like this article because it is well written and easy to follow.

For me, I think the usage of APTES is cool and I should remember this for future work as well as the fabrication of microtubule roads using photolithography.