Bradley Scott Colton and Dr. Marc Hansen, Physiology and Developmental Biology
Overview
The purpose of our experiment was to prove the interactions between three different proteins named nectin, zyxin, and VASP. It is thought that these proteins are involved in forming interactions with the actin cytoskeleton between neighboring cells and add to the cell adhesion between cells in an epithelial layer of cells. Normally cells need to stick together to function together as a layer of cells. One of the characteristics of cancer is when cells break these cell-to-cell adhesions in order to migrate to different areas of the body and set up new tumor sites. This process is known as epithelialmesenchymal transition.
Our goal was to investigate this process on a molecular level. Our lab had previously shown that epithelial cells that had intact zyxin-mediated actin connections would not separate when induced to undergo EMT. However the way that zyxin-actin interaction is connected to the cell membrane is still unknown. Our hypothesis is that zyxin anchors to sites of cell-cell adhesion via binding to nectin.
Results
In our first experiment we proved the interaction between zyxin and nectin. We used recombinant DNA techniques to isolate purified zyxin and nectin. Then ran put the isolated protein and one sample with both proteins in a sucrose density gradient centrifugation to show. Our results showed that the same with both proteins, both zyxin and nectin showed up in the same fraction at a different fraction than then the isolated protein controls. This proves that zyxin and nectin do interact.
In the next step of our experiment we looked to build on the knowledge we gained from the above experiment and prove how the nectin-zyxin complex interacts with actin via binding VASP. We proved this interaction by running a series of actin pull-down assays with all possible combinations of nectin, zyxin, and VASP with actin. In this experiment the actin sinks to the bottom of the tube forming a pellet. Any protein that binds to the actin or another protein already bound to the actin will be found in the pellet. In our results we showed that with all proteins present, they are all found in the pellet. Using result from the different combinations, we also were able to prove how this interaction takes place (as shown above in figure 1). We proved this by showing that zyxin only is found in the pellet with actin when VASP is present, and nectin is only found in the actin pellet when both VASP and zyxin are in the sample.
Complications
Our experiments did not do just as smoothly as we had predicted and delayed our finishing our project by a couple months. The route of our problems was on the molecular biology level. The plasmid we were trying to make to produce the nectin we needed to isolate and use in our experiments was just not working. We thought we had solved the problem and continued with our experiments until we got some unexpected results in our sucrose gradient experiment. All of our tests with nectin were not working. We then ran some tests and found that the protein we had isolated was not functional nectin. We were eventually able to solve this problem by ordering a plasmid that was effective in making function nectin.
Future
Although we had some set backs in our experiments our expected outcomes are the same. We are currently working on compiling our data and putting together all of our results in a presentable format. We can put together a poster that we can submit to present at the American Society for Cell Biology. We are also using our results combined with other results from our lab to put together a journal paper characterizing the role of these proteins in EMT that we can submit to several different journals. Once we finish writing up or results and now that we have effectively proven that that zyxin anchors to sites of cell-cell adhesion via binding to nectin, we will start looking at other aspects of EMT. We have already talked about looking more closely at role of several other proteins that have showed a change in expression levels during the process of EMT. Hopefully, as we can continue to research the different proteins that contribute to metastasis, we can piece together how they interact with each other and get closer to being able to develop potential cancer treatments.