User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/06/04/Movement of microtubules by single kinesin molecules

From OpenWetWare

Jump to: navigation, search

Contents

Disclaimer

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

Paper

Movement of microtubules by single kinesin molecules

Review

  • They want to know more about chemomechanical transduction....fancy words for "they are interested in how molecular motors move". They are wanting to look at:
    • Forces produced from single motors.
    • The distances one of these motors moves when it hydrolyzes ATP.
    • Transition rates between the power stroke and the binding of ATP.
  • Ultimately, they want to make an experiment that has low numbers of kinesin molecules attached to a glass coverslip and observe microtubule motility. They claim to be able to determine from this:
    1. Attachment time for a kinesin molecule to grab a hold of a microtubule.
    2. Forces under which a kinesin molecule can work.
    3. Magnitude of displacement produced during 1 ATP hydrolysis.
  • Their experiment was to adsorb kinesin on glass and then look at microtubules moving along them with dark field microscopy.
  • They state in the introduction that nothing occurred until a minimal concentration (100 nM) of kinesin adsorbed onto the glass was able to move microtubules. The good thing is that they also state that they didn't use anything to coat the glass. They suspect that this is why you need such a large concentration of kinesin.
  • The tubulin polymerization buffer they used is:
    • 4 mM MgCl2
    • 1 mM GTP
    • 10% DMSO so the Taxol in solution they add later will go into solution
  • Their "standard" buffer is
    • 80 mM PIPES
    • 1 mM EGTA
    • 2 mM MgCl2
    • pH 6.85 adjusted with KOH
  • They did do an experiment where they pretreated the surface of the coverslip. Their pretreatment was: tubulin with 4 µm cytochrome c. They also tried 15 µM haemoglobin.
  • After finding that a single kinesin motor can move a microtubule, they also determined the following:
    • At saturating amounts of ATP (1 mM) the speed of microtubules was independent of kinesin density.
      • This means that a single kinesin molecule can move a microtubule as fast as a whole bunch of them. Go Kiney!
    • At very low densities of kinesin, microtubule speed was independent of it's length.
  • They mention that the speed of microtubules follows the Michaelis-Menten equation for low kinesin densities and low ATP concentrations.
  • They note that when there is a lot of kinesin around, microtubule speed decreased. Thus they concluded that microtubule speed at low ATP concentrations is actually a function of kinesin density and not ATP concentrations.
    • This is a good analysis. They say that the reason microtubules go slower when lots of kinesin are attached to them is because of ATP. Imagine two kinesin holding onto a microtubule. Now imagine that only one gets and ATP so it goes through a power stroke. The microtubule will buckle between the kinesin but it won't move until the other kinesin gets an ATP and can go through a power stroke. Very nice.
  • They estimate that a kinesin produces forces above 60 fN.
  • Looks like they put an upper bound on the step size of kinesin from their measurements of 15 - 30 nm.
  • Ha! They state the "hand over hand" motion.

Take home

  • Lots of information.
  • They determined that one single kinesin molecule can move a microtubule. I think this is the most important fact for me.
  • Historically, they put an upper bound on kinesin's step size.
  • They showed that you have to coat glass with something so that the kinesin don't denature. The idea of denaturing is not confirmed but, I know from hindsight that this is the case.
  • Over all, it was a good article but, the beginning had a little too much pompous vocabulary for my taste. Plus, their sentence structures in the article was overly complex in my opinion and they didn't veer from this prescription very often. I did learn a lot and I am very glad to have read it.
Personal tools