User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/06/04/Taxol stabilization of mitotic spindle microtubules - analysis using calcium induced depolymerization

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Contents

Disclaimer

This is my review not your review. Sorry, that was rude. Don't read my notes until you read the paper please.

Paper

Taxol stabilization of mitotic spindle microtubules: analysis using calcium-induced depolymerization

Review

  • Goal - To report on the rate, extent and reversibility of taxol stabilized microtubules. They used microtubule spindles from sand dollars.
  • They state that taxol does not affect the assembly of actin.
  • Taxol apparently also does not compete with the binding of microtubules associated proteins.
  • The experiment was to use 100 µM Ca2+ to analyze the rate, extent and reversibility of stabilization of microtubules with taxol.
  • They state that DMSO at 0.1% in solution or less does not affect experiments. No data was shown for this statement.
  • They used sand dollar microtubules and in the process of getting those microtubules they used a "lysis" buffer which is deceptively similar to BRB80...
    • 5 mM EGTA
    • 0.5 mM MgCl2
    • 20 mM PIPES
    • 15% glycerol (v/v)
    • 1% Nonidet P40 - I'm guessing their detergent.
    • pH 6.8
  • Found that 1 µM and 10 µM taxol stabilized microtubules but they did not "fix" them completely.
  • They found that it takes time for the taxol to bind to the microtubules.
    • Question: What is the on rate for taxol?
  • Note: This paper keeps using the initials BR for birefringent retardation and they use a very similar buffer to BRB80. Could the BRB stand for birefringent retardation buffer? The world will never know.
  • This is great. They show that stabilization of microtubules with 10 µM taxol is ideal for quick stabilization. The literature says to use 10 µM taxol but, they never said why.
  • They don't know at this time where taxol binds to the microtubules.
  • They note that 100 µM taxol is way less than the amount needed to induce depolymerization for in vivo brain microtubules (mM).

Take home

  • Use 10 µM taxol to stabilize microtubules. Taxol does not fix microtubules.
  • Even after the treatment of taxol to microtubules, they can still depolymerize.
  • Great article and an easy, if long, read.
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