Simple is better

Today was a more science filled day. We wanted to see if partitioning in polymerizable monomers would increase the stability of an ooctyes plasma membrane. The plasma membrane is held together by weak van der waals interactions, and something as simple as transferring a piece of membrane across an air-water interface is enough to disturb the bilayer structure. If we want to use ICCR's in a biosensor platform, we need to satisfy several criterion, two of which our lipids research hopefully fulfills: we must 1) provide a hydrophobic environment that, for the most part, mimics the protein-lipid interactions found in a real plasma membrane such that the ICCR correctly folds, and 2) provide a membrane with extended lifetime for longer term use in, e.g. diagnostics or high throughput screening of drugs.

In an ooctye, a large amount of structural stability is provided by the vitelline membrane. In our experiments, we decided to remove this membrane and directly partition monomers into the plasma membrane. That part, and the polymerization was easy, but, the big question was, how would we quantitatively determine whether the oocyte had increased structural stability?

Generally, ooctyes have a spherical shape, which is relatively easy to deform, especially once the vitelline membrane (VM) is removed. If you were to transfer the VM-less oocyte to a paper towel, the surrounding water would quickly soak up, and the oocyte would flatten out like a pancake. In contrast, if the VM is still attached, the oocyte still has some structure even after the surrounding water is removed. To give an idea of it's shape, picture a water droplet you've put on your desk; it's somewhat round, but not completely. We could use this physical test to observe structural stability, comparing non-peeled ooctyes, peeled oocytes, and polymerized oocytes. Alternatively, we could try chemical tests, exposing oocytes to different solvents.

Christophe and I tried both physical and chemical tests. To dry the surrounding water, we used a piece of paper and a paper towel. The paper was much slower at absorbing water, so we discontinued its use. The towel was useful. It absorbed water quickly. However, it was hard to discern the oocyte shape. From there, we moved onto chemical tests. We tried ethanol, soapy water, decane, SDS solutions, and chloroform. The chloroform was funny, because it immediately turned the low density polyethylene petri dish into a goopy mess. As a chemist, I should have known that would happen, but in the spirit of investigation, I was mostly caught up in testing the stability.

Suprisingly, the oocyte can handle many chemical insults. They withstood ethanol; chloroorm and decane removed some of the lipids on the dark side of the oocyte, but did not compromise its stability; SDS and soapy water caused the oocytes to disintegrate, but only after an extended period (20 minutes). Certainly these tests should be performed again to ensure we obtain the same results.

From there, we decided that an air-water interface transfer provided the easiest to interpret results, it was simply a matter of how to replicate the transfer without the need of a towel. Christophe took some oocytes sitting in a petridish of water and removed the water with a pipette. Voila!! The oocytes behaved similar to the paper towel; the peeled oocytes flattened like a pancake and the non-peeled oocytes kept their shape. The polymerized oocytes were a bit harder to discern, and we'll try again. In the end, the best test was the simpliest.