User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/12/01/Taxol Crystals Can Masquerade as Stabilized Microtubules

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Contents

Paper

Taxol Crystals Can Masquerade as Stabilized Microtubules

Review

Great paper describing how tubulin will form on crystalline structures of Taxol. This is great. This describes how I was able to get this structure from my data. It makes complete sense. Microtubules are floppy when in solution. This structure is totally rigid which could mean that I have tubulin forming on a Taxol crystal.

I would like to note what this group calls BRB80.

  • 80 mM PIPES
  • 1 mM EGTA
  • 2.1 mM MgCl2
  • 1 mM GTP
  • 4% Glycerol

This is different from what I use but oh well.

SJK 01:37, 2 December 2009 (EST)
01:37, 2 December 2009 (EST)I think that BRB80 almost always refers to the buffer without the osmotic stress agent (glycerol or DMSO) that is added.  So, I'd say these authors are a bit out of the norm.  I remember this from our saga on FriendFeed searching for the origin of the name BRB80 (I'm still leaning towards "Borisy reassembly buffer," but Brinkley is fine too). (BTW: Your friendfeed thread is the #1 hit I got on google search for brb80).  Here's two examples of old papers where BRB80 is defined without the glycerol: PMID 3248521 (Kellog, Mitchison, Alberts "Behaviour of microtubules and actin filaments in living Drosophila embryos.") and the kinesin home page: http://www.proweb.org/kinesin/Methods/motility.html
01:37, 2 December 2009 (EST)
I think that BRB80 almost always refers to the buffer without the osmotic stress agent (glycerol or DMSO) that is added. So, I'd say these authors are a bit out of the norm. I remember this from our saga on FriendFeed searching for the origin of the name BRB80 (I'm still leaning towards "Borisy reassembly buffer," but Brinkley is fine too). (BTW: Your friendfeed thread is the #1 hit I got on google search for brb80). Here's two examples of old papers where BRB80 is defined without the glycerol: PMID 3248521 (Kellog, Mitchison, Alberts "Behaviour of microtubules and actin filaments in living Drosophila embryos.") and the kinesin home page: http://www.proweb.org/kinesin/Methods/motility.html


Questions

So if Taxol makes these crystal structures, what happens when Taxol is in D20? Does it crystallize? Could this be where I'm getting the "log" structures in my data?

Finally, are the ribbon structures that form when microtubules are polymerized in heavy water be from the crystallization of Taxol?

SJK 01:00, 2 December 2009 (EST)
01:00, 2 December 2009 (EST)This could potentially be partially addressed by trying to visualize MTs w/o taxol.  You can polymerize them in light water and then (maybe) stabilize them by diluting into D2O.  If not too expensive, polymerization in GTP analogs could also work (such as GMPCPP in that other paper you linked).
01:00, 2 December 2009 (EST)
This could potentially be partially addressed by trying to visualize MTs w/o taxol. You can polymerize them in light water and then (maybe) stabilize them by diluting into D2O.

If not too expensive, polymerization in GTP analogs could also work (such as GMPCPP in that other paper you linked).
SJK 01:23, 2 December 2009 (EST)
01:23, 2 December 2009 (EST)Oh, and now I see your "log" comment.  I'd say for big logs yes, probably.  However, my intuition is that the MT-MT sticking that you were seeing is not from taxol microneedle crystals.  To me, those looked like native MTs, in which case the taxol binding site should be on the inside.  But it's definitely worth trying this at zero taxol concentration (if possible) and if not possible, markedly reduced taxol concentration.  One thing we probably won't know is the solubility of taxol in D2O.  Wow!  Complicated!
01:23, 2 December 2009 (EST)
Oh, and now I see your "log" comment. I'd say for big logs yes, probably. However, my intuition is that the MT-MT sticking that you were seeing is not from taxol microneedle crystals. To me, those looked like native MTs, in which case the taxol binding site should be on the inside. But it's definitely worth trying this at zero taxol concentration (if possible) and if not possible, markedly reduced taxol concentration. One thing we probably won't know is the solubility of taxol in D2O. Wow! Complicated!


PLOS One structure

So the basic structure of this paper is,

  • Intro & statement of purpose.
  • Results and justification of purpose.
  • Materials and methods.
SJK 01:20, 2 December 2009 (EST)
01:20, 2 December 2009 (EST)Awesome find on this paper!  I have definitely seen those structures in the past.  I have also seen things I called "logs"--definitely thicker than normal MTs, but rod-like with little branching or aster-formations.  I wonder if this is what they're referring to as a "crystalline needle?"  One other thing that I noticed is that these "log" or aster structures had much much much less photobleaching.  Would that mean the rhodamine labels are trapped between the taxol crystal and the tubulin?  Or at least some of them are?  I haven't read the entire paper yet to see if they've commented on this.I am really glad you found this paper, for two reasons.  First, it answers a mystery and can substantially improve our assays.  Second, I think it's a very good model for the PLoS paper you were discussing today: to me it seems to convey a similar kind of story.
01:20, 2 December 2009 (EST)
Awesome find on this paper! I have definitely seen those structures in the past. I have also seen things I called "logs"--definitely thicker than normal MTs, but rod-like with little branching or aster-formations. I wonder if this is what they're referring to as a "crystalline needle?" One other thing that I noticed is that these "log" or aster structures had much much much less photobleaching. Would that mean the rhodamine labels are trapped between the taxol crystal and the tubulin? Or at least some of them are? I haven't read the entire paper yet to see if they've commented on this.

I am really glad you found this paper, for two reasons. First, it answers a mystery and can substantially improve our assays. Second, I think it's a very good model for the PLoS paper you were discussing today: to me it seems to convey a similar kind of story.
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