User:Andy Maloney/Kinesin & Microtubule Page/Microtubule papers/Kinesin follows the microtubules protofilament axis

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Disclaimer

These are my notes on the following article. Please read the article before reading my notes.

Paper

Kinesin follows the microtubule's protofilament axis

Notes

  • They found that kinesin walks along MT protofilaments.
    • They were able to visualize twists in the MTs with electron cryo-microscopy.
  • A 12 protofilament MT has a right-handed supertwist pitch of 3 to 4 µm.
  • A 14 protofilament microtubule has a left-handed supertwist pitch of ~6µm.
  • They used bovine brain tubulin that they purified.
  • They used kinesin from the bovine brain and purified it themselves.
  • They used 3 buffers.
    • BRB80
    • 80 mM PIPES
    • 1 mM MgCl2
    • 1 mM EGTA
    • pH 6.8 with KOH
    • MES buffer
    • 100 mM MES
    • 1 mM MgCl2
    • 1 mM EGTA
    • pH 6.5 with HCl
    • Phosphate buffer
    • 10 mM sodium phosphate
    • pH 7
  • They used all kinds of buffers for polymerization.
    • Axoneme doublets
    • PIPES-Glycerol
      • PIPES + 36% glycerol
    • PIPES-DMSO
      • PIPES + 5% DMSO
    • PIPES
    • MES-Glycerol
      • MES + 36% glycerol
    • MES-DMSO
      • MES + 5% DMSO
    • Phosphate buffer + 2 µM Taxol
  • They state that the supertwist can be predicted by the "Lattice-Rotation Model".
  • They passivated their glass with ~2.5 mg/mL casein.
  • They used our antifade solution.
  • Yeah! VHS!
  • They used something called MEASURE hardware from Walsh Electronics and software from Block to measure velocity.
  • We polymerize MTs in PIPES-Glycerol. This means that we should have about 50% of the polymerized MTs with 14 protofilaments and about 40% with 13. The remaining 10% is in the 12 or 15 protofilament class.
    • There is a difference between what we do and what they did. They first initially grow MTs in PIPES + Glycerol to obtain seeds. They then finish polymerization in just PIPES. They also polymerized in 36% glycerol + PIPES. We polymerize in PIPES + 6% (v/v) glycerol so we may not have the quoted ratio of 14mers and 13mers.
  • They say that doublet-seeded MTs had 90% of the polymerized MTs with 13 protofilaments.
  • They were able to detect rotation by a "tail" on the MTs. This tail is an incomplete MT with fewer protofilaments. It was dimmer than the rest of the MT and I have seen this in my data but, only rarely. I think this "tail" is an artifact of how they were purifying their tubulin and their polymerization.

Above is a false colored image of the MT with a tail. Up in the left hand corner. False coloring was done with ImageJ. You will have to click on the full resolution image to see the tail.

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Take home

It would appear to me that during polymerization of MTs, you will get a spread of MTs with different protofilaments. This spread depends on what you are polymerizing the MTs in.

  • I wonder if it depends on the osmotic pressure?
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