User:Brian P. Josey/Notebook/2009/09/10

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GTP Magnesium Salt and Questions Andy had me look for GTP magnesium salt, but I could not find any. We decide that since we need the magnesium in solution, we could use buffers with a little bit of magnesium chloride, MgCl2, in solution. We were also talking about why we using GTP in the first place. Essentially, when the tubulin dimers polymerize into the microtubules GDP rest in the center of one of the dimers. Another one comes to the side, and some how it uses magnesium ions to create a chain of tubulin dimers creating the microtubule. I asked a couple of questions, and I'm leaving them here so I have a reference somewhere that includes my questions about what we are doing. How does the tubulin use magnesium? Does the concentration of magnesium decrease? How fast do the tubulin polymerize into microtubules? Why does the kinesin go toward the positive end? I have an idea on some of these, but I'm going to do some more research into them to see what I can find out, and maybe find a topic or two to investigate on my own. Steve Koch 22:39, 10 September 2009 (EDT): Great questions, Brian! I don't have good answers for the first two, which I would combine into: "Does magnesium get incorporated into microtubules?" My guess is "yes," since tubulin catalyzes GTP hydrolysis and thus I'd expect it to need Mg++ for that. You can find my kinesin / microtubule saved PDFs on the controller and in there, I have a whole slew of PDFs I've saved which address the issue of GTP use in MT polymerization. (Andy can show you how to find these.) From what I remember, each stable tubulin dimer has a GTP in there more or less permanently. Whereas when two dimers polymerize, another GTP binds between the two dimers. The GTP is hydrolyzed at some point, resulting in a less stable tetramer (aka dimer of dimers or dimer-dimer). As for "how fast," that too has been studied a lot. It's not straight forward, since it's a nucleation/growth process that's highly concentration dependent. Furthermore, osmotic stress agents strongly affect the growth (we use glycerol). And of course, temperature and other buffer properties (ionic strength) strongly affect process. PEM80 (developed by Borisy--Ask Andy) was developed to be sort of optimal for at least some kind of tubulin prep. There are also some really good studies of single-microtubule polymerization rate. Finally, "why does kinesin go toward the positive end" is a classic question. ncd is a kinesin that actually walks towards minus end. Some great classic papers mixing and matching parts from ncd and "conventional kinesin" (now called kinesin-1) to see which parts determine directionality. When I'm around, let me know and I'll show you some papers. Or you can just dive into my library. Or, you can also check out my citeulike library to see what I've read recently. For the past 6 months or so, I've tried to put short comments there when I store the article. You can see my comments using the green callout icon.