User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/04/10/Kinesin & Microtubules

Paper
Equilibrium and Transition between Single- and Double-Headed Binding of Kinesin as Revealed by Single-Molecule Mechanics

Review
I'll try again with this paper. My previous attempt at reviewing it was terrible and I said some rather nasty things. I'm pretty sure I was in an all around bad mood that day and I apologize for being so harsh. The paper has generated a lot of questions that I think are good ones. Perhaps more knowledge in this subject will teach me the answers. Or better yet, some of my questions may generate papers themselves.

Introduction

 * Tubulin subunits are approximately 8 nm in length. This is why kinesin takes 8 nm steps. It steps between tubulin subunits.
 * Each step requires 1 ATP of fuel.
 * The length a kinesin will walk (run length) along a microtubule depends on
 * The ionic strength of the buffer.
 * Temperature.
 * The amount of fixing the microtubules.
 * Question: Does the run length depend on pH? What about if you change the water to heavy water?


 * I still need to commit to memory what ATP hydrolysis is.
 * Kinesin likes to walk in the "+" direction along a microtubule.
 * They claim that kinesin primarily binds only one head to microtubules if ATP is not present in solution but ADP and AMP-PNP are.
 * They claim that kinesin primarily binds both head to microtubules if just AMP-PNP is present and no ATP.
 * Question: So they are talking about kinesin motility in two different buffers. What is the current model for kinesin motility?


 * They claim that the average unbinding force depends on the loading direction, i.e. in the +/- directions of the microtubules.
 * They claim that the apparent ratio between single and double head binding depends on the loading rate.
 * They claim to be able to resolve when a double head binding turns into a single head binding on microtubules. Just for the AMP-PNP buffer. They can do it by looking at the elastic modulus of kinesin during loading. They claim the transition rate is 1 1/s.
 * Complete kinesin detachment from microtubules can occur for both single and double head binding.
 * They claim that the double headed binding state of kinesin is predominant under equilibrium in the absence of applying a load for both their buffers.
 * They claim that the lower the loading rate, the higher the probability for single headed unbinding.

Materials & Methods

 * Their buffers are made up of
 * 2 mM MgCl2
 * 80 mM PIPES-KOH
 * 1 mM EGTA
 * pH 6.8
 * 4.5 mg/mL Glucose
 * 0.22 mg/mL Glucose oxidase
 * 0.036 mg/mL Catalase
 * and they differ by the inclusion of either


 * I thought personally that it was cool that you can purchase microtubules that are fluorescently labeled differently by their "+" or "-" ends. Not sure how you would go about doing this though.
 * Question: Why would they use EGTA to chelate out Mg when they use MgCl2 as their primary source of ions?
 * They note that kinesin is unstable in ATP free buffers with no microtubules present.
 * Question: I don't understand why then they call their first buffer "nucleotide free". If kinesin is unstable in ATP free buffers, then how are they able to do the experiments?
 * Question: Does Amylase take a long time to scavenge all the ATP? If it does then there is ATP in solution when they were doing these experiments.
 * Question: Does Amylase act very quickly to get rid of ATP? If it does, then that means kinesin has residual energy stores available so it can continue to stay bound to the microtubules.
 * Question: If kinesin does not have energy stores to stay on the microtubules, then this means that kinesin wants to bind to the microtubules and it is the ATP that helps remove a kinesin head from the microtubules so it can take a step. Which way is it? Is it the ATP that "glues" kinesin to microtubules or is it the ATP that removes kinesin from the microtubules?
 * Question: Why would kinesin be stable when it is attached to a microtubule when there is no ATP, but isn't when there are no microtubules and no ATP? Is it stable when there is ATP and no microtubules?