User:Andy Maloney/Kinesin & Microtubule Page/Microtubule papers/Kinesin follows the microtubules protofilament axis
These are my notes on the following article. Please read the article before reading my 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.
- 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 + 36% glycerol
- PIPES + 5% DMSO
- MES + 36% glycerol
- 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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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?