User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/05/11/Mechanism of Microtubule Stabilization by Doublecortin review

Disclaimer

This is my review. Not yours. So please don't take my review as meaning anything until you have read the paper. Plus, I may be wrong with my thoughts. If I am, please feel free to correct me.

Paper

Mechanism of Microtubule Stabilization by Doublecortin

Review

• Doublecortin (DCX) binds between protofilaments.
• DCX preferentially binds to 13 protofilament microtubules. They show data that says over 70% of microtubules polymerized with DCX in the sauce have 13 protofilaments.
  • Note: This is great to know because if we want to make reproducible microtubules in the lab, we may want to use DCX.
• They used a diffractometer. I think this is just a meter to detect scattering.
• Figure 1 of the paper has some very interesting things.
  • Question: Why are the microtubules with DCX added smaller?
  • Question: Why would the microtubules with DCX be more ordered? That's fighting entropy! What's going on here?
    • Steve Koch 14:46, 11 May 2009 (EDT): entropy fighting doesn't bother me at all--there's plenty of energy around here (consider how ordered the MTs themself are!) The fact that +DCX look more ordered could be a MT size effect. It also could have to do with the MTs being more 13-protofilament type. 13-protofilament MTs have straight PFs, so I could believe that this would cause stronger MT-MT interactions (though i'm just making that up). I would say the fact that the ends appear to be aligned would argue against DCX cross-linking MTs in their middles. (That they're lined up indicates to me a weaker interaction, but all along the MTs, allow it to relax this preferred configuration.) Lined-up ends could also indicate DCX crosslinking of specifially MT ends. It would be interesting to know whether these MTs are parallel or anti-parallel. And whether they are more likely to bundle in bulk solution.

• Unfortunately their logic for stating that DCX helps tubulin polymerize into microtubules isn't very clear and I'm not sure their data represents this statement. Their logic is: because adding DCX to tubulin makes the microtubules smaller, it must mean DCX helps create nucleation sites for microtubule growth and this is why the microtubules are small. They didn't check to see if there was the same amount of tubulin in each sample, those with DCX and those without. Plus, their logic is circular.
  • Steve Koch 14:41, 11 May 2009 (EDT): I don't see any kind of circular logic. Plus, they do start with the same tubulin concentration. The only thing I see missing is a count of MT number--so, they can see shorter MTs, but I don't see whether they specifically say that they see "lots of short MTs." This may be hard to do with cryo-EM (overall counting), but should be easy with light microscopy. I think the nucleation hypothesis is more likely than a mechanism for DCX stopping polymerization after 1 micron.
    • Andy Maloney 18:00, 11 May 2009 (EDT): I see your point. My statement is a case of me not knowing enough. I guess I just didn't agree with their wording because it wasn't clear to me.
• They state that 13 protofilament microtubules do not make helical structures.
  • Question: What type of conformation does a 13 protofilament microtubule make?
• They state that DCX does not overlap kinesin binding sites. They don't show any data but I think this statement is referenced in the article.
• They state that tubulin bound to GDP undergoes a curving in its conformation and this is why GDP-tubulin will want to cause catastrophes for microtubules.
  • They reference this statement. The reference is here.
• They also state that Taxol binds on the inner wall of microtubules. Thus Kiney can walk all the way without being stopped by a Taxol molecule.
  • They again reference this statement with the reference here.

Take Home

• The article is overall clear and precise except for their circular logic for DCX helping nucleation sites. (Update See above for clarification.) Since they were mainly talking about microtubule stabilization using DCX, this is a minor problem. I would have liked to see time series data where they show how well DCX stabilizes microtubules but their high resolution diffraction experiments are nice.

• They don't give you what you would expect from the title, a measure of DCX's stabilizing properties.

• They reference statements quite nicely. Props for this!

• Steve Koch 14:48, 11 May 2009 (EDT): It would sure be nice to have more 13-protofilament MTs in our assays! It's also possible that these shorter MTs would be ideal for tracking, etc. On the other hand, having another protein (DCX) around to worry about certainly makes things more difficult and may complicate analysis and comparison to others' work.

Doublecortin

First, this group references things really well. I can only hope that when I go to write papers, I can be at least 10% as efficient with referencing things as they are. In fact, I am going to make it my mission to reference as well as they do. Thanks Koch for showing me their papers.

At any rate, you can't buy doublecortin. I did a quick search for papers and it appears you can recombinantly grow DCX. I'm a little frustrated with this. Mainly because I don't know how to do it and I really want to know so if I need something, I can make a bug grow it for me. Hmm, I guess this is why biophysics is tough. You just can't buy everything you need.