# User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/03/17/Kinesin & Microtubules

## Paper

Here is a link to the paper: Kinesin Movement on Glutaraldehyde-Fixed Microtubules.

## Basics

Before moving on to my review, I need to define some terminology that I was initially unfamiliar with in the article.

In this paper, the authors discuss a better method for fixing microtubules by using a chemical that cross-links it. Not being a biologist, I had no clue as to what this word cross-linking meant. Well, in the context of microtubules, cross-linking means using a chemical that strengthens the bond between subunits. As I found out in the last paper, Nanomechanics of Microtubules, microtubules are weakest between protofilaments and thus are prone to dissociate at those bonds. Using a chemical, glutaraldehyde helps to stabilize microtubules so you can experiment with them.

### Hydrolyze/Phosporalation

Also in this paper they mention kinesin and dynein hydrolyzing ATP. The chemical definition of hydrolysis is when water is broken into its constituents namely,

H2O $\leftrightharpoons$ H++OH-

Now they talk about kinesin hydrolyzing ATP. I'm assuming they mean ATP $\rightarrow$ ADP in this process. Somehow in this chemical reaction, a water molecule is broken up and a phosphate is lost. I'm going to chalk this up to nomenclature because I do not know the chemistry behind calling this reaction such.(Steve Koch:This part of the ATP article may help)

Of course, phosphorylation is when something gets a phosphate stuck to it (so ADP $\rightarrow$ ATP). I'm not a proponent of Orwell's 1984 Newspeak but come on... When ATP $\rightarrow$ ADP, ATP loses a phosphate and thus is dephosphorylated. There must be a reason as to why chemists and biologists call the above reaction hydrolysis.

### Oligomerization

Oligomerization is a fancy word that means subunits of polymers that get stuck together in small numbers.

### Remember chemistry?

I've forgotten some of these words and I am going to put them here again to remind myself. I especially like Wikipedia's definition of an amine group. I quote it below.

amine - Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Their picture of this group is even better.

### Basics questions

I am pretty confused with some of these questions. I've never done these type of chemistry procedures before so I don't understand why you would want to do these things.

• Why would you use sucrose to cushion what you are centrifuging?
• Why would a chemical called Tris stop cross-linking?
• Steve Koch 13:01, 18 March 2009 (EDT): Man, I learned a lot from reading your notebook! I learned that Tris is short for "Trisamine" and that it has an amine group. Thus, if you add a shitload of tris to the cross-linking reaction, it's going to react with the carboxyl groups on the glutaraldehyde and thus quench the reaction. I don't know why I've never seen this before. Whenever I've use the carboxyl to amine linking (such as with the EDC protocol), we used glycine to quench the reaction.

## Review

• They comment that microtubules and their subunits, tubulin, are in a dynamic equilibrium in the cytoplasm.
• They note that the turnover rate for tubulin in microtubules is approximately 15 minutes in vivo. I'm not sure how they determine this, but it may be something good to know. Now in in vitro studies, microtubules do not exist in dynamic equilibrium with tubulin because no know has figured out how to stabilize them with tubulin in solution.
• Since we cannot stabilize microtubules with tubulin in solution, experimenters have turned to chemicals to prevent microtubule destabilization. Previous to this study, they use a drug called Taxol.
• I have no idea what this means but they say it: They note that the most probable reaction of cross-linking due to glutaraldehyde may occur from cross-linking the primary amine groups of lysine residues on the microtubules. Of course they don't say where the lysine residues are on the microtubules.
• They give a procedure for preparing microtubules on glass.

Take home

This paper was just littered with nomenclature that I was unfamiliar with. I really need to start practicing my chemistry terms and learn more biology when it comes to kinesin and microtubules. At any rate, the take away messages include:

• Microtubules fixed with glutaraldehyde lasted longer than those fixed solely with Taxol.
• Kinesin did not loose its affinity to bind to glutaraldehyde fixed microtubules as long as the amount of fixing was low. They did not quantitatively measure kinesin's affinity change though.
• They state that cross-linking may occur in tubulin and not between protofilaments.
• Kinesin's on/off rates were affected by glutaraldehyde cross-linking. They stayed on fixed microtubules for less amount of time than non fixed microtubules.
• Kinesin's motility was not affected by glutaraldehyde cross-linked microtubules.