Cellular biologists are generally concerned with four major cellular processes: mitosis, apoptosis, DNA methylation, and DNA replication. The aim of my project is to design a tool to allow researchers to interrogate these four major cellular processes in a living cell. Such a tool will have valuable implications for research on a wide range of diseases from cancer to Parkinson’s.
The basis for this tool is a special type of tiny antibody found in camelids (alpacas, camels, llamas, etc.), known as the nanobody. We are working with nanobodies that bind to four different antigens endogenous to the cell: actin, lamin, proliferating cell nuclear antigen (PCNA), and Dnmt1. Each nanobody is also fused to one of four different fluorescent proteins: mTFP (teal), mKO2 (orange), EYFP (yellow), and tagBFP (blue). By fusing a different fluorescent protein to each of our four nanobodies, we are able to image (via a confocal microscope) these antibodies as they bind to their target antigens within the cell.
The logical question here is “So what?” Of course, its cool to look at a cell that’s glowing four different colors, but why is this important to science? Well, as I mentioned in my intro to this post, apoptosis, mitosis, DNA methylation, and DNA replication are the four major cellular processes that cellular biologists are concerned about. As it turns out, each of our fluorescently tagged nanobodies, which I will now refer to as chromobodies, tell us something about each of the four major cellular processes. Our actin chromobody binds with actin, a major component of the cell’s cytoskeleton, thus allowing us to characterize the cell’s shape and structure. Our lamin chromobody binds with lamin, a component of the nuclear lamina which encloses the cell nucleus, allowing us to visualize the cell’s nucleus. Dnmt1 is responsible for DNA methylation, therefore our Dnmt1 chromobody allows us to see where transcription of the cell’s genome is being turned off due to methylation. Finally PCNA is a molecule that must be present for successful DNA replication, so our PCNA chromobody allows us to see when a cell is replicating its genome.
Thus, our four chromobodies can effectively act as a toolkit allowing us to interrogate a cell about its activities. Kind of neat, right? Right now we are creating plasmids containing the sequences of our chromobodies (via Gateway Cloning) and then transfecting cells with them. Once we have live cells expressing our chromobodies, we can look at look at these cells with a confocal microscope and ‘ask’ them a series of questions. Is this cell getting ready to die? Is this cell getting ready to divide? What are the patterns of DNA methylation? The answers to these questions are particularly pertinent to the Caron Lab’s main research goals. These goals include better understanding how G-protein coupled receptors (GPCRs) mediate the development of disease.
And this is only the beginning. The next step will be to take this technology and apply it to cancer research. The long term goal is to fuse four different fluorescent proteins to a wild-type oncogene and three of its mutations. We can determine which of the four genes are randomly expressed (in a mouse model) based on the coloring of the cells. From there we can compare cells within the same animal model to see the varying effects of different oncogene mutations on the fate of a cell. Note that this description of the lab’s long-term research goal is a basic, shallow summary. Right now I will not go into depth about the methods and mechanisms required for such a project. Here I am merely giving a general, plainly-worded summary of our goals. Just three weeks ago I had no clue this kind of thing could even be possible. I find it thrilling that I’m getting to play even the tiniest role in this larger body of exciting new research.