User:Andy Maloney/Kinesin & Microtubule Page/Surface passivation/How calcium causes microtubule depolymerization

Disclaimer

These are my notes on this paper. Please read the paper before reading my notes.

Paper

How calcium causes microtubule depolymerization

Notes

There are lots of good and interesting things in this paper. I will list most of them below.

• Experiment
  • They used calcium to trigger catastrophes in microtubules. Catastrophes mean that microtubules shrink. Not sure why this terminology is used.
  • Two types of microtubules were investigated

1. phophocellulose-purified (PC) microtubules
2. Microtubules with microtubule associated protein (MAPs) on them.

• They show that calcium induces the disassembly of microtubules for both PC and MAP microtubules.
• They showed that rescue rates reduced when the temperature of the microtubule solution decreased. Rescue is when microtubules start to grow again.
• Question: Will we need to get a temperature stabilizer for our experiments?
• Question: This paper shows that free calcium will affect microtubules. What about calcium affecting microtubules fixed with Taxol?
• They showed that above 500 µM of free calcium, catastrophe rates of microtubules were enough to make them shrink.
• They also note that free magnesium will cause catastrophe as well. But, it starts at 5 mM.
• Note: Lots of buffers I've seen people use include using MgCl2 and Casein. Casein has calcium phosphate in it and as I just found out, magnesium will cause catastrophe as well. This is probably why people use EGTA/EDTA in their buffers.
  • Question: Is there a specific reason as to why people use MgCl2 in their buffers? Does either kinesin or microtubules need a little bit of it to work? Yes! Apparently someone has asked this question and this paper talks about it. Magnesium actually aids in microtubule growth. (Steve Koch 00:07, 20 January 2010 (EST): Mg++ cofactor for ATP hydrolysis also. w/ GTP for polymerization. ATP for motility)
• They note that MAP microtubules are more stable and exhibit better contrast in their DIC images.
• Of course, the most interesting part of the paper doesn't have data showing this. Apparently they saw MAP microtubules stop all rescue/catastrophe events after about 20 - 30 minutes at 37˚C. The microtubules were even stable with the addition of mM concentrations of calcium. Whether or not this statement is true needs to be investigated.
• They talk a lot about GTP stabilization and GTP caps on microtubules.
  • Note: Should I be concerned about knowing what this means?
  • (Steve Koch 00:11, 20 January 2010 (EST):I can't get this paper either. Without looking, I think what they're referring to is that the MTs ends are more stable when GTP is between dimers. When actively polymerizing, there is a GTP cap, because polymerization is outpacing GTP hydrolysis. I'm not sure what the latest on this is, though.

Take home

Calcium affects microtubules. Casein has calcium in it. People use EGTA/EDTA to stop calcium from screwing with microtubules but, does calcium screw with microtubules fixed with Taxol?

Good paper.