Jellyfish and Glowing Proteins

By: Erica Gorenberg

From the stars on the ceiling of a childhood bedroom to the key chains brought home as souvenirs, some of the most memorable trinkets from youth are those that glow in the dark. For molecular biologists, like for kids, the ability to harness fluorescence and make molecules reminiscent of those glow-in-the-dark toys was one of the most useful and exciting innovations in modern science.

Green fluorescent protein (GFP) was first observed in 1962 in a bioluminescent jellyfish, Aequeorea victoria, as the molecule responsible for the animal’s ability to glow. The protein was isolated, and researchers demonstrated its ability to light up green under beams of specific colors of light. The use of GFP revolutionized biological research–in the time since GFP’s discovery and use, molecular biology has entered its golden age.

Osamu Shimomura, who first isolated the protein, Martin Chalfie, who first used GFP to track other proteins, and Roger Tsien, who discovered the properties that make GFP fluorescent and manipulated them to create a rainbow of fluorescence proteins, were awarded the 2008 Nobel Prize in Chemistry. Before their work with GFP, visualizing proteins within live cells was far more complex, and less dependable, as it relied on the insertion of fluorescent dyes into the cell where they could bind to the proteins of interest. These dyes were unreliable because they weren’t always specific to their proteins, and because the physiology of the cell had to be disrupted to add them.

Now, through the use of DNA modification technologies, the gene for GFP can be fused to genes of other proteins, allowing proteins to be produced with the fluorescent molecule attached. This is called "tagging" the protein with GFP. As Martin Chalfie demonstrated for the first time in 1994, GFP can be used to show specific proteins and cellular structures in living organisms, providing researchers with new insights into cellular function. For example, when the GFP gene is attached to the gene for a microtubule associated protein, MAP2, one molecule of GFP is produced and fused with every molecule of MAP2. By viewing cells that produce MAP2, such as neurons as shown in Figure 1, researchers can learn how much of the protein is expressed under different conditions, simply by measuring the intensity of GFP’s fluorescence. The more MAP2 that’s produced, the brighter the GFP signal will be.

Figure 1. Hippocampal neuron stained with GFP for MAP2. Actin filaments are in red and DNA is in blue. The protein tags allow researchers to see MAP2 in the neuron’s long projections, or dendrites, and see that actin localizes in clumps along the dendrites at structures called spines. The DNA stain shows the cell’s nucleus.   Image via Halpain lab at UCSD  .

Figure 1. Hippocampal neuron stained with GFP for MAP2. Actin filaments are in red and DNA is in blue. The protein tags allow researchers to see MAP2 in the neuron’s long projections, or dendrites, and see that actin localizes in clumps along the dendrites at structures called spines. The DNA stain shows the cell’s nucleus. Image via Halpain lab at UCSD.

With a GFP tag, researchers can also see where the protein is made, where it is transported, and under what conditions this changes. Previously cells had to be fixed, killing them and freezing them in time to view their components. One of the most compelling aspects of fused fluorescent tags is their ability to be viewed in real time within living cells. Live imaging allows researchers to manipulate the cells and understand how different environmental changes can affect cellular components over time. By using different colors of fluorescent proteins to label different cellular proteins and looking at where and when they overlap with one another, researchers can look within the cell to understand how different proteins might interact.

Within each tagged cell is a constellation of glowing proteins, like the glow-in-the-dark stars stuck to a childhood ceiling. These cellular constellations move and interact in breathtaking ways that scientists are only beginning to understand, thanks to the discovery of GFP.

What makes mad cows mad: The story of prions

By: Erica Gorenberg

In the years following the first human case of “Mad Cow Disease” or variant Creutzfeldt-Jakob Disease (vCJD), world governments introduced measures meant to prevent the infection of additional animals and to protect humans from the continued spread of the disease.

Diseases like mad cow, or bovine spongiform encephalopathy (BSE) in cows, had been documented in animals and humans throughout the world long before the 2003 outbreak. In humans, Creutzfeldt-Jakob Disease (CJD) was first described in 1920, and Kuru, “the laughing sickness” was discovered in the Fore tribe of Paupa New Guinea in the 1950s. In sheep, the equivalent disease is known as Scrapie, because as the disease progresses, the sheep scrape themselves against anything they can find, causing severe injuries. Although these diseases had been studied for many years, it wasn’t until the the 1980s that researchers understood that, unlike previously known infectious agents like bacteria or virus, these diseases were caused by an infecting protein, also known as a prion.

            Each of the thousands of proteins made in a cell has a specific sequence of amino acid building blocks that denotes how it should fold in order to function properly. Most cells in the human body make PrP, the protein that can cause CJD and the other prion diseases mentioned above, but in unaffected individuals it is harmless. In contrast to the normal form of PrP, its prion variant, PrPSc, has a conformation that is harmful to the cell and that can take the normally-folded version and convert it into the infectious misfolded version. Basically prions are the bad kids that your parents didn’t want you to hang out with in high school.

As if prion proteins weren’t already causing enough damage, PrPSc clumps together, inhibiting the normal function of the cells. When too much protein aggregation occurs, cells activate a suicide pathway, known as apoptosis, in order to prevent the spread of harmful materials by breaking them down. Under normal circumstances, misfolded proteins are broken down, but prion aggregates are resistant to the cell’s normal protein breakdown system, the proteasome. In prion disease, more and more cells die, leaving brain tissue porous and spongy and contributing to the symptoms of the disease. In humans, CJD and Kuru manifest first with dysfunctions in muscle coordination and progress rapidly to include personality changes, memory impairment, dementia and eventually death.

Prion proteins usually infect their hosts through consumption or contact with contaminated material. Only in rare cases do sporadic genetic mutations in the PrP gene lead to heritable prion disease. It seems BSE spread to cows because the protein in their feed came from scrapie-infected sheep. When humans consumed infected cow meat, the prion proteins of the cows were similar enough to pass along PrP misfolding to their human counterparts, creating vCJD.

The prion hypothesis has been controversial since its proposal, but more and more research stands to support the idea of infectious proteins. Now, researchers are able to purify PrP and study animal models that are helping them to understand how this protein may first spontaneously misfold to cause the diseases. Many questions remain unanswered, and a cure for prion disease has yet to be found, but research in this field continues. To understand prion disease, we must learn if PrP, even in its prion form, may exist to aid the cell in some way and whether diseases like Alzheimer’s or depression may be caused by prion-like proteins.


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