User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/10/19/Lipids discussion
So I am going to start a discussion on my reasons as to why I have chosen the lipids I have for surface passivation. There are several reasons why I want to try different types of lipids and I think an open discussion about what I know about them is warranted.
I should also say that I want what I write below to actually mean something more than just a notebook entry. I want this to evolve into either a paper or a chapter in my dissertation.
The basic structure of a lipid can be quite complex. However, the lipids I am interested in for surface passivation are of the phospholipid group. A phospholipid is amphiphilic in nature where amphi means both or on both sides and philic means to be attracted to. So an amphiphile is a term giving to substances that like to be in both hydrophobic and hydrophilic environments.
The basic structure of a phospholipid is given in the image below.
The two major components are the hydrophilic head group and the hydrophobic tail groups. When phospholipids are introduced to an aqueous environment, they will self aggregate into multilayer structures that have the tail groups excluding water and the head groups wanting to be in an aqueous environment. This amphiphilic nature of phospholipids is beneficial for biological systems because it allows for cellular wall structures.
Not all amphiphiles are phospholipids. In the case of surface passivation for gliding motility assays, we use a protein called casein to passivate the surface of a glass slide. Casein is not a phospholipid, however, this protein does exhibit amphiphilic behaviors. In mammals, calcium is essential for life and is especially crucial for growing infants. Evolution has create a vehicle to transport calcium phosphate to infants and that vehicle is the casein protein. Calcium phosphate is hydrophobic so it will not go into an aqueous solution and thus a vehicle is needed to deliver it to an infant.
There are problems associated with using casein for kinesin and microtubule assays. The major reason for not wanting to use casien is the calcium phosphate it transports. Calcium is known to depolymerize microtubules so using a substance that has calcium in its structure can cause problems for kinesin and microtubule experiments. To circumvent the possibility that calcium will leach into solution and cause problems for the microtubules, people add a small amount of EGTA to their buffers. EGTA is a metal chelator that has a high affinity to chelate calcium from solution. While this is a great idea, no one has asked how EGTA may affect kinesin's processivity in the experiments. People have studied the influence of EGTA for microtubule polymerization and they found that it does basically nothing.
Armed with the knowledge that casein transports calcium phosphate and that calcium depolymerizes microtubules, I have been studying ways to passivate glass surfaces with phospholipids in order to completely eliminate the need for using casein and thus the need to use EGTA in the buffers. Another reason why I want to eliminate EGTA from the solution is based on another experiment that I want to do that involves changing the osmotic pressure of kinesin and microtubule assays. Completely getting rid of the EGTA in solution may change the osmotic pressure drastically and thus cause kinesin to no longer walk along microtubules.
Lipids under investigation
So far I have used two different types of phospholipids as surface passivators. One is called phosphatidylcholine (PC) and the other is phosphatidylethanolamine that has a polyethylene glycol polymer attached to its head group (PE-PEG).
PC is a phospholipid purified from chicken eggs. The type I purchased came as a mixture of differing lengths of hydrocarbon chains with both saturated and unsaturated hydrocarbon tails. This fact is important because the length of the hydrocarbon chain tells you what the transition temperature is for the lipid membrane to go from a gel phase to a fluid phase. In general, the smaller the hydrocarbon chain, the lower the melting temperature is. I should note that this applies to saturated hydrocarbon chains only. In general, if the chain is unsaturated, it will melt at very low temperatures.
I believe that the transition temperature is very important when trying to use lipids as a surface passivator. The reason I believe this is because of the nature of a fluid membrane. The lipid molecules in a membrane are free to move a considerable amount when in the fluid phase. Now if a kinesin is bound to the lipid membrane and is attached to a microtubule, there is nothing preventing the membrane from putting stress to the microtubule and thus causing it to shear enough till it rips apart. Thus, I believe that it is essential to get a membrane that has a melting temperature far beyond room temperature.
The other lipid, PE-PEG, does have a melting temperature above room temperature so there is a possibility of using it as a passivator. The major obstacle preventing me from using this lipid effectively as a surface passivator is the PEG polymer attached to the PE head group. Originally I thought that I needed a different lipid molecule to help passivate the surface so I chose one with PEG on it. Unfortunately the PEG molecule is so long that there is no chance that a microtubule will get close enough to the surface to visualize motility. They all stay in solution. Even more of a disappointment is that if a kinesin is stuck to the end of the PEG molecule and is attached to a microtubule, there is no way I will observe motility because of the floppy nature of the PEG polymer.
New lipid approach
After investigating PC and PE-PEG, I have come to the conclusion that I have not thought through the usage of phospholipids as surface passivators enough. So, after considerable thought, I have come up with a plan that uses different lipids.
My initial thought process started with melting temperatures for lipids. I am again using a PE phospholipid but this time it does not have any PEG on it. There are 18 carbon atoms in the hydrocarbon chains and this lipid has a melting temperature around 70˚C. I believe that this is hot enough to combat the room temperature and the heat from the mercury lamp during observation. I am hopeful that I will not see the same sort of ripping apart of microtubules that I saw using PC.
PE is going to be my base lipid. What I mean by this is that the membrane I will create will have mostly PE in it because of its high melting temperature. I will then dope the PE membrane with other lipids to see if I can get kinesin to stick to the membrane. I plan on doping the membrane with 2 different types of lipids. Both of which are made up of 16 carbon saturated chains. The difference between the doping lipids are going to be the charge on the head groups. One will have a positive charge on the head group and the other has a negative charge.
The reason for trying charged head group lipids is based solely on trying to investigate why casein works so well as a passivator. The simplest thing to ask is; Is there a charge on the casein that kinesin likes to stick to? If there is, then having a charged head group will help to investigate this question.
I want to reiterate what my ultimate goal is. I want to find a different surface passivator to run my kinesin and microtubule experiments on. This way I can get rid of the need for EGTA in solution.