User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/04/23/The role of casein in supporting the operation of surface bound kinesin

Disclaimer

This is my interpretation of the below paper. Please do not take my review as a "Cliff's Notes" to the paper. These are my thoughts and if you disagree, I welcome feedback on the talk page.

Paper

Super awesome kick ass open access paper!

• The role of casein in supporting the operation of surface bound kinesin

Review

• They state that kinesin denatures when it is not supported on glass.
  • Question: Why would this be the case? Does silicon or oxygen do something funny to kinesin?
  • Note: They do reference this paper about kinesin denaturing on glass.
• They state that kinesin exert a maximal force of ~ 6 pN and are about 50% efficient.
  • Note: I'm not sure what they mean by this statement. Are they talking about the amount of force necessary to hinder the motion of a kinesin along a microtubule?
• Casein is included in the motor and microtubule solutions based on a qualitative observation that it improves microtubule motility.
  • Oh yeah, they are speaking my language.
• Casein has 4 different subunits ranging from 20 - 30 kDa. They are
  • [math]\displaystyle{ \alpha }[/math]_s1 (12%–15%), two distinct hydrophobic domains
  • [math]\displaystyle{ \alpha }[/math]_s2 (3%–4%), two distinct hydrophobic domains
  • [math]\displaystyle{ \beta }[/math] (9%–11%), one distinct hydrophilic and hydrophobic domain
  • [math]\displaystyle{ \kappa }[/math] (2%–4%), one distinct hydrophilic and hydrophobic domain

and exist in (%) quantities for cow milk.

• The goal
  • To look at microtubule motility experiments by changing the type of casein (whole, [math]\displaystyle{ \alpha }[/math], [math]\displaystyle{ \beta }[/math], or [math]\displaystyle{ \kappa }[/math]) blocking the glass substrate.
• Finally someone tells me what BRB80 buffer is

1 mM MgCl₂

1 mM EGTA

80 mM PIPES

ph 6.9

• Casein preparation

1. Add casein to buffer
2. Centrifuge and remove the top portion of undissolved casein. I know there is a word for this, but it excapes me right now. I don't know why this step is necessary.
3. Filtered through a 220 nm filter.

• • Question: Hmm, how big are casein molecules? I'm assuming they are doing this because they don't want micelle structures that are too large. How big do casein aggregates get?
• Note: So my whole excursion on casein was based on the question Why do we need EGTA in the buffer?. I found out that casein is the primary vehicle for delivering calcium and phosphates to infant mammals. But, calcium depolymerizes unstabilized microtubules. (I still don't know about Taxol stabilized ones.) I thought that since EGTA chelates magnesium and calcium, this is why we use it. In actuality, we use casein because it prevents kinesin from adsorbing on glass and then denaturing thus destroying the experiments. From what I've read so far, it seems that using EGTA is because everyone uses the legacy buffer BRB80. I'm not sure though it it still warrants investigation.
• The results

• • This table shows the results of their experiments. The take home message from this data is
    • If you don't block the glass with casein, you won't get any microtubules.
    • If you put casein in with the kinesin to coat the glass, then you will get microtubules.
    • If you include casein when you add microtubules, you will get more microtubules supported by the kinesin.
• They state that they think casein is making a bilayer on the surface of the glass and thus prevents kinesin from touching glass and denaturing.
  • Note: I guess it's good that they said this but it seems obvious to me that this would happen since casein's subunits are amphiphilic. Maybe people think casein is more stable as micelles on the glass and this is why they said this.
• They state that [math]\displaystyle{ \beta }[/math] casein is the best at supporting microtubule motility and that [math]\displaystyle{ \kappa }[/math] casein is the worst. They make these measures by looking at how many microtubules are in solution > 1 µm in length and moved for greater than 2 µm.
  • Note: The microtubules in the [math]\displaystyle{ \kappa }[/math] casein experiment were all greater than 2 µm and there were always a few of them.

I would say that the best images were from the [math]\displaystyle{ \kappa }[/math] casein assay. Plus, this looks like the best picture to analyze with programs. This is awesome! This paper actually answered a question of mine.

Take home

Use of EGTA in buffers may be a legacy issue and I need to look more into this. Also, I was wrong about why we want to use EGTA. Casein is used to prevent kinesin denaturing due to interactions with the glass. Due to the use of casein, I thought we needed the EGTA to chelate excess calcium to prevent depolymerization of the microtubules. But, it turns out we need casein for kinesin and not the microtubules.

You need to coat glass so that it won't denature kinesin. What you coat it with may not need to be casein but it is what everyone uses. Blind use or not, I need to make sure I can do what people have done before I do something else.

It seems that [math]\displaystyle{ \kappa }[/math] casein is the way to go for our experiments. Sigma sells [math]\displaystyle{ \alpha }[/math] casein, [math]\displaystyle{ \kappa }[/math] casein, and [math]\displaystyle{ \beta }[/math] casein but, we should just get the [math]\displaystyle{ \kappa }[/math] casein. Oddly enough, you can recombinantly grow cow [math]\displaystyle{ \kappa }[/math] casein in E. coli. That is messed up and so freaking cool at the same time. Hopefully Ant will be able to do this soon.

Some final thoughts are, why not use a pegylated lipid to do the job of casein? Ant and I were talking about this earlier today and it seems like it is something we should try. I have experience with lipids and making liposomes and bilayers so it will be super easy to do. Plus, a lipid is a simple thing compared to casein. Just a thought. Maybe the group that did this paper is trying it.

• IK: Kinesin might not bind to glass properly through a lipid bilayer. Actually it's quite amazing that it works with casein as well as it does. But of course worth a try.
• Steve Koch 22:47, 23 June 2009 (EDT): I agree about the amazing part w/ casein. One thing we're thinking is that for sure kinesin can attach to lipid bilayers in real cells. But whether we can accomplish that w/ recombinant kinesin is another issue (maybe light chains are required, etc.). But since Andy has the lipids expertise, it's definitely worth trying.