User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/04/20/Microtubule paper review

Casein

What is casein? It appears everybody uses it to coat coverslips to prevent microtubules and other stuff from adsorbing onto parts of the coverslips you don't want. But, why do people use it? What are the advantages? What are the disadvantages? I'm going to jot down some ideas about casein and why I think there is a better method. This idea will go in my "To Do" experiments.

What is casein?

Casein is a globular protein that people believe forms micelles. It is primarily found in milk products and it appears that it is a vehicle for calcium phosphates. Calcium phosphate is insoluble in water (i.e. non-polar) so it can be delivered via micelles since the inside of micelles are non-polar. I think evolution needed to find a way to deliver calcium and phosphates to growing mammals and this is why casein is predominantly found in milk. You can also find it in those buff mixes Ant and Larry drink after working out. Eww, by the way.

Using casein as a coating for coverslips in microtubule experiments automatically sends red flags to me. Why? Well, it appears that microtubules have a trigger to decrease in size when calcium is present in solution, see below paper. To get around this, people use either EGTA or EDTA to chelate calcium from solution.

I found an article by D. Schmidt where he describes how casein changes, due to chelating calcium. I'm in the process of obtaining the article from the library and hopefully it will answer a lot of my questions when it comes to casein and calcium.

Why do people use casein?

From what I can tell, people use casein because it works. Someone found out that it works and everybody started using it. Whether or not it is the best solution, I do not know. As far as I can tell, this is what is going on with casein.

People adsorb tubulin and then grow microtubules on coverslips or they adsorb Taxol prepared microtubules on coverslips. They then use Casein to cover all the glass that does not have microtubules on it. Why then does Casein not allow microtubule growth or adsorption on those areas covered by it?

I think that there are two reasons why microtubules will not grow on areas covered by Casein, and one of them is entropy. As I mentioned above, Casein is a globular protein. As such, it has an entropic desire to remain globular. If you stick a microtubule on the glob, then where the microtubule is stuck, that area is no longer an entropic glob. So, reducing the entropy of the glob gives rise to a force that resists the reduced entropy and thus a microtubule is repelled by the glob.

The second reason I think casein is used is because it has calcium ions in it. As the below paper reveals and what is known in the field, calcium ions actually cause microtubules to depolymerize. This is a good and a bad thing. If we want microtubules to grow and stabilize on our coverslip, we need to not have them grow every where. So, in a sense, adding casein prevents the growth of new microtubules in our system by effectively making a sea of poisonous goo around the microtubules that are already fixed to our coverslip.

If a microtubule actually overcomes the entropic force repelling it from the casein, it will ultimately end up depolymerizing due to the release of calcium in solution.

With that said, how much calcium ions in solution does it take to depolymerize microtubules? Something like 500 nM. How much calcium phosphate is in casein? Something like 7 mM! That's an order of magnitude difference. Plus, it seems like it would be really difficult to actually determine the amount of calcium phosphate in casein (as is evident from the paper I got the 7 mM figure from, review to come later).

The physicist in me wants to propose we make our own micelles either from SDS or Pluronic loaded with a known quantity of calcium phosphate. That way we know how much calcium is in solution. We could even make liposomes loaded with a known amount calcium chloride.

Casein's advantages and disadvantages

The obvious advantage to using Casein is that is a proven method used to coat coverslips and prevent microtubules from growing or adsorbing onto the glass where the Casein is. Is it the best way? I'm not sure. Actually, I don't think so.

My knowledge on casein is limited but it appears that when you use it, you don't know how much calcium may be in solution. With that said, this means you need to use another chemical to chelate the calcium out of solution. People do this by adding either EGTA or EDTA in the several mM range to the solution. So what. If it works then who care?

Koch question: One of our goals is to look at how kinesin moves along microtubules. The important thing about our project is that we are interested in how kinesin's movements will change dependent on the addition of osmolytes. Are EGTA or EDTA going to be considered osmolytes?

If it works, but screws with what we are doing, should we be using it? Or, should we get a handle on what people have done before and then once I've gotten some experience, should we try doing what a physicist would call "cleaner" experiment?

Steve Koch 23:39, 20 April 2009 (EDT): Yes, any solute is going to exert osmotic stress (a.k.a., change the water chemical potential, a.k.a., change the water activity, a.k.a., change the osmotic pressure). However, "good" osmolytes are going to be neutral. EDTA is highly charged in solution, right? Therefore, it's going to affect the ionic strength of the solution, and that's going to have a bigger effect on things and at an earlier concentration. You can see really dramatic differences from ionic strength going from 10 mM to 100 mM to 500 mM. Whereas from what I've seen, 200 milliosmolal is sort of the minimum needed to see an effect on protein-DNA intereactions. (I'm not being precise here.) I guess what I'm trying to say is that if you have enough charged solute to exert an osmotic stress, then you're going to have a very strong ionic effect as well. But you SHOULD be aware of the osmotic stress from ionic species, something that I didn't consider with my EcoRI salt studies (and which many people before me didn't mention either). Another thing, when your solute dissociates (such as disodium EDTA), you have to count all of the independent ions created as separate. E.g., dissolving 100 millimol / liter of disodium EDTA would be somewhere around 300 milliosmolal...I'm sort-of guessing, though, because I don't have good handle on the terminology yet. Finally: measuring the actual osmotic pressure is going to be key. Evan is bringing us a vapor pressure osmometer, wahoo!

Andy Maloney 01:20, 21 April 2009 (EDT): So many things here. First some terminology.

1. Osmolality has units of (osmoles / kg of solvent). I remember the difference between solute and solvent by the first letter in the words that are different, u and v respectively. Since u (letter 21) comes before v (letter 22) in the alphabet, the term solute talks about the smaller substance in a solution. So, salt water would have the solute as salt and the solvent as water.
2. Osmolarity is a concentration. (osmoles / L of solution). I like to talk about the osmolarity of a substance because it is similar to the molarity (as opposed to the molality). I think it's easier for a physicist to think about concentration as opposed to a number with inverse mass units.
3. As for counting the species in solution to find the osmolarity, not only do you have to take into consideration the number of EDTA molecules that have calcium bound to them, but you also have to take into consideration the number of EDTA molecules that do have calcium bound to them.

[math]\displaystyle{ \mathrm{EDTA}_{\mathrm{free}}+\mathrm{Ca}_{\mathrm{free}}\leftrightharpoons \mathrm{CaEDTA} }[/math]

The [math]\displaystyle{ \leftrightharpoons }[/math] is there for a reason. EDTA binds calcium in a reversible manner. This means that there is a [math]\displaystyle{ K_d }[/math] for EDTA (an attraction for calcium). You can make pretty good guesses on the number of free versus bound calcium ions are in solution as well as the number of free and bound EDTA molecules. This is in fact how you calibrate solutions with known calcium concentrations in them. But, if you are wanting to calculate the osmolytes in solution, you need to know how to calculate bound versus free EDTA molecules because as you said, a free EDTA molecule will contribute more to the ionic strength of a solution.

1. Now, as you said, you don't see differences until you get around 200 mOsm. Well, people put into solution around 1 mM EGTA/EDTA so I guess we won't have to worry about it.
2. From what I can tell, microtubules will undergo a catastrophe when it comes in contact with calcium. The only source of calcium in every solution I've seen is from Casein. Unless people are using hard water.

I guess my issue is that no one has told me why I should use Casein when I know using it will release calcium ions into solution. To prevent the calcium from tearing apart my microtubules I have to use another chemical, EDTA/EGTA. I don't like taking a drug for a symptom of another drug. But, I'm happy to conform to legacy crap for the beginning experiments. I just hope someone reads this and can tell me more.

• One final thing, making polydisperse micelles or liposomes is easy! If we want to coat a coverslip with them instead of magical Casein. Then we don't have calcium in the system and kinesin gets its desired amphiphiles in solution.