How do cancer cells invade tissues and metastasize? How do proteins play a role in muscle cell contractions that control our heartbeat? How does the Plasmodia parasite infect our blood cells to cause malaria?
Our understanding of health and disease have made enormous strides, in part because of the power of fluorescent proteins. Thanks to these colorful proteins, scientists can now visualize the biological processes that control the healthy functioning of our bodies - and give us insight into how these processes go wrong in disease.
What is Fluorescence?
Fluorescence is the phenomenon of a substance emitting visible light after absorbing non-visible light, such as ultraviolet light. For example, fluorescent light bulbs contain two key ingredients: mercury and calcium chlorophosphate. When a fluorescent light bulb is turned on, the electricity energizes the mercury atoms, which give off ultraviolet light. This ultraviolet light is then absorbed by the calcium chlorophosphate, a fluorescent compound. After the UV light is absorbed, the calcium chlorophosphate emits the visible light that we see.
To our surprise, fluorescence isn’t only found in light bulbs!
Discovering Fluorescent Proteins
The first fluorescent protein was discovered in the jellyfish Aequorea victoria, which is found off the west coast of North America. Green Fluorescent Protein (or GFP, as it’s colloquially called) was discovered by Dr. Osamu Shimamura and its properties were further studied by Dr. Roger Y. Tsien. Dr. Martin Chalfie then pioneered the technique of adding GFP to specific parts of DNA. This allowed him (and now countless scientists all over the world) to make specific cell types fluoresce green! All three scientists shared the Nobel Prize in Chemistry in 2008 for their discovery and application of GFP.
The Many Uses of Fluorescent Proteins
Normally, (most) cells and proteins are invisible to us. Adding fluorescent proteins to a specific cell type - like a certain type of brain cell or immune cell - and using special fluorescent microscopes allows us to easily visualize their shape and location in the body. Adding fluorescent color to specific proteins allows us to see where in cells or tissues those proteins are found at any given time. This helps us understand biological processes and gives us clues to the functions of proteins and cells in human health and disease.
While GFP is the most widely used and well-known fluorescent protein, there are now many different fluorescent proteins used today, in many different colors. By combining different fluorescent proteins, scientists can study how proteins interact, how cells are shaped, or even how different cells are interconnected to dictate the morphology of entire tissues. Check out the different cell types of the retina, for example. You can see retinal ganglion cells (RGCs, labeled with red fluorescent protein), amacrine and horizontal cells (ACs, labeled with green fluorescent protein) and photoreceptors and bipolar cells (PRs, labeled with cyan fluorescent protein).
Expressing Fluorescent Proteins in Tissues
This is all possible because of our understanding of gene expression systems. While every cell in our body has the same DNA, each cell type expresses different genes which underlies what sets them apart from all the other cell types. Scientists have taken advantage of the systems that regulate gene expression in order to differentially visualize cells with fluorescent proteins.
So what does it take to express a gene? In all of our cells, gene expression is regulated by many factors but TLDR let’s talk about one crucial component: the promoter. Not all of our DNA is made up of genes - in fact, almost 95% of our DNA doesn’t encode a protein. Promoters are one such example of ‘non-coding’ DNA and they play an important role in regulating if and when a gene is expressed.
This means that if we added DNA that contains only a gene to a cell, the cell would very likely not express the encoded protein. Why? Because it is lacking the promoter - the bit of DNA that helps give the cell the instructions of when and where to express the gene.
Here’s where it gets interesting: each different type of cell has its own unique profile of promoters. This allows muscle cells to express the precise genes at the right time and amounts needed to perform the functions of a muscle cell - while neurons, having their own set of unique promoters, can express the exact combination of proteins to carry out the functions of our nervous system.
So, if scientists want to express a fluorescent protein in a specific cell, they attach the corresponding promoter that regulates gene expression in that cell. For example, in the retina, the Atoh7 promoter is known to express genes in retinal ganglion cells, while the Ptf1a promoter regulates gene expression in amacrine and horizontal cells. Similarly, the Crx promoter is involved in gene regulation in photoreceptors and bipolar cells.
By cutting and pasting the GFP gene from the jellyfish into a piece of DNA that also includes a specific promoter, scientists can control in what cells the GFP gene is expressed. The implications of this technique has transformed how we visualize cellular processes and with this, has advanced our understanding of health and disease processes including cancer, HIV and malaria.