Imaging DNA and Lamin A Protein Using AFM

Eukaryotic cells are composed of tens of thousands of genes, neatly organized and packed in the cell’s nucleus. The genome is structured in chromosomes, each occupying a specific nuclear volume, known as chromosome territories. Proper cellular functions like gene regulation and cell mitosis rely on adequate chromosome organization. Hence, understanding the mechanisms responsible for the organization of chromosomes, and, more specifically, key proteins which are involved in the process, is important to further investigates such functions. In recent study conducted in our laboratory, it was shown that chromosomal inter-chain interactions formed by lamin A protein throughout the nucleus, contribute to chromatin dynamics. Moreover, the critical role of lamin A in maintaining the genome organization was suggested. The objective of the present study was to investigate the interaction of DNA molecules and lamin A proteins under in-vitro conditions. For this purpose, I used Atomic Force Microscopy (AFM) to investigate the configurations of “naked” DNA compared with DNA that was interacting with lamin A. DNA with lamin A was more difficult to measure compared to "naked" DNA. It took us several attempts until we set up a suitable protocol and got an image of the DNA with lamin A. The images show that "naked" DNA seems unfolded and separated, while DNA with lamin A are flexible and tend to do loops. In addition, it was found that lamin A can link together many DNA molecules. Our challenge is finding the optimal concentration of the protein, so that its interaction with the DNA will be visible, but it will not cause too much protein on the surface. In addition, we observed that the protein, like DNA molecules, is negatively charged. Therefore, high concentration of it disturbs the system from easily conducting the measurements. This, as well, implicates that the levels of protein cannot be too high.