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.
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?