Roger Tsien and the GFP story: from its discovery to the latest applications.

In 2016, the Nobel prize winner Roger Tsien passed away, shaking the scientific world. Roger Tsien received a Nobel prize in Chemistry in 2008 with Osamu Shimomura e Martin Chalfie, for the discovery and development of the green fluorescent protein, GFP, as a new research tool that revolutionised the study of cell biology.

GFP is a protein that shines once hit by blue light. Today, thanks to the studies of Dr Tsien, researchers can choose among a vast array of coloured fluorescent proteins, enabling the labeling of different proteins at the same time, in living cells and whole animals.

 

The Green Fluorescent Protein.

GFP was isolated from the jellyfish Aequora Victoria, also called Crystal jelly. Aequora Victoria is a jellyfish common on the west coast of north America. Almost completely transparent, it emits a blue/greenish light. This property of jellyfishes, fireflies, or some microorganisms including bacteria, to emit light as a consequence of some chemical reactions is very common and fascinated scientists. In the 50s and 60s, biochemical methods were commonly used to isolate the proteins responsible for this naturally occurring chemiluminescence.

Fluorescence in Aequora Victoria is due to two proteins: Aequorin and GFP. Aequorin is made of two subunits. Between these subunits there is a chromophore (see Glossary): Luciferin. The chemical reaction following the binding of calcium ions to the chromophore pushes electrons from an excited state to a resting state, with the emission of energy as light. The emitted light, in turn, excites GFP that, fluorescing, confers the greenish aspect to the jellyfish.

  

Using fluorescent proteins in single cells.

Fluorescence was already widely used in cell biology, by immunolabeling proteins of interest with specific antibodies (see: https://archive-sciencewhatelse.jimdo.com/immunolabeling/). However, the use of fluorescent antibodies has a major limitation: it can only be used in fixed cells and tissues.

In the early 90s, the cloning and expression of GFP in exogenous cells opened the way for many applications: GFP can be fused to other proteins, enabling to follow their movements within the cell and analyse the associated biological processes, in living cells, in real time.

Fluorescent proteins have multiple applications, such as:

 

- the visualization of protein Trafficking, which means the movement of a protein from one cell compartment to another one. One example is the protein called CLIC1, or chloride ion channel 1. CLIC1 is normally expressed in the cytoplasm of several cell types. In case of cell stress, CLIC1 moves from the cytoplasm to the plasma membrane. CLIC1 movement to the plasma membrane has been demonstrated in microglia cells both by using antibodies in fixed cells, and by tracking CLIC1-GFP movement in living cells.

 

- Chromophore activated light inactivation (CALI) is a quite recent technique and it is based on a by-product of fluorescent proteins. Indeed, fluorescence has a “side effect”, causing the production of Reactive Oxygen Species, ROS, which are harmful for the cell. For this reason, when acquiring images in live cells, scientists tend to reduce intensity or exposure time of the cells to the light, in order to avoid cell damage. With CALI, ROS produced by the reaction of fluorescence are actively used. KillerRed is a fluorescent protein modified to be particularly efficient in producing ROS upon light stimulation. ROS production following light stimulation will damage the proteins in tight contact with KillerRed. Damaged proteins will stop functioning, so the cell signalling and the cell process linked, will be silenced.

CALI-mediated inactivation is more specific than the very popular silencing with RNA interference (see methods section of this website for details on RNA interference technique: knock down) for several reasons.

Firstly, ROS produced by a single light stimulation are active only for a short time, as the cellular ROS scavenging system (see Glossary) will soon remove them. So, a specific protein and related process can be shut off only for a short period. Through an accurate control of light stimulation, silencing can be obtained with high temporal precision.

Secondly, with CALI, protein functionality can be controlled also with high spatial precision. This is important as some proteins have different roles in different cell compartments. For instance, the protein kinase PKA (see Glossary) is expressed both in nucleus and cytoplasm. In the cytoplasm of cardiomyocytes, PKA activation modifies, by adding a chemical group (phosphorylation), MyBP (Myosin binding protein), a protein with a -still unknown- role in muscle structure and function; while activated PKA in the nucleus phosphorylates the protein CREB, ultimately affecting gene expression. The expression of KillerRed targeted to the cytoplasm, would switch off its activity, without affecting the nuclear isoform, enabling the study of the cell process solely linked to the cytoplasmic PKA.

 

- 2-FRET and 3-FRET to check protein interaction, each of them fused to a fluorescent protein. The principles of FRET can be checked on this video https://archive-sciencewhatelse.jimdo.com/what-is-fret/ . Briefly, when a protein X is labeled with a fluorescent protein (eg CFP) and another protein Y is labeled with another fluorescent protein (eg YFP), if the two proteins X and Y will get close enough to each other’s during a specific cell process, there will be a FRET signal. More recently, this technique has been improved: in 3-FRET, another protein, we will call it Z, can be labeled with a different fluorescent protein (eg mRFP). If X, Y and Z are close enough, they will interact in a trimeric complex. Upon light stimulation, CFP emission will excite YFP that in turn will excite mRFP. FRET is also commonly used to measure the dynamics of molecules and cyclic nucleotides. cAMP is a second messenger involved in several cell processes, and FRET sensors have been developed to measure cAMP dynamics. For instance, the sequence of a cAMP binding protein is fused between a CFP and a YFP. The conformational change following the binding of cAMP gets CFP and YFP far away from each other’s, leading to a change of fluorescence signal. Measurements of fluorescence signal will mirror the changes in cAMP concentration (see details on the video https://archive-sciencewhatelse.jimdo.com/what-is-fret/  ).

 

Applications

Recently, fluorescent proteins have been used to label tumor cells. A fluorescent protein has been fused to a positively charged residue, normally used to drive proteins inside a cell, and a negatively charged sequence, which prevents, instead, the entry inside the cell. Tumors overexpress some proteins called proteases that are involved in degradating some parts of the extracellular membrane in order to metastasize. When the fusion protein is in proximity of tumor cells, it will be cut by proteases: the negatively charged sequence will be released, freeing the positive sequence that will now drive the fluorescent protein inside the cell.

The experiments from Tsien lab have shown that with this method the uptake of the fluorescent protein is higher (2-3 times higher) in tumor cells (expressing the proteases) compared to the healthy surrounding tissue.

One of the main challenges in cancer treatments today is the impossibility to specifically recognize and target cancer cells over the healthy ones. This method opens the way to a future selective targeting of therapeutic agents in tumor cells over non-tumor cells, or a specific labelling of tumor cells over healthy tissue in surgery. Indeed, one of the main way to treat solid cancer today is the surgical removal. However, despite the sophisticated technology associated with surgery, surgeons have to distinguish each different tissue (nerves, cartilage, fat, muscles, blood vessels,…) by relying mainly on their colour, varying in different shades ranging from white to red. Fluorescent probes targeted to specific tissues can provide a colour-code for the surgeon, to clearly distinguish each tissue and avoid to damage, for instance, a nerve during a tumour removal, ameliorating the outcome for patient’s health after surgery. At the same time, specifically labeling tumor cells, can help the surgeon in identifying residual cancer cells even if covered by other tissue.

 

Conclusions and comments

A group of scientists, fascinated by the properties of a jellyfish, years later revolutionised the study of cell biology. Probably even they could not figure how their discovery would have affected scientific research fifty years later. Sometimes is hard to understand where basic research could lead us. The GFP story is only one example of the importance and the power of curiosity-driven basic research, to remind us of its importance in parallel to applied research. We never know where this is going to lead.

However, sometimes is fun to stop and take our time to think about a possible future application of our research. Maybe we will do it on day, maybe someone else will, but it is anyway a good exercise for our mind, and there is nothing as important in science and research than keep our brain always on.

 

Glossary:

Chromophore: Part of a molecule, or a protein, that due to the chemical bonds between its atoms, confers colour to the protein.

Ros scavenging system. ROS are a normal byproduct of some cell processes. However, while low doses are not only tolerated but represent a signal of specific cell processes (e.g. a peak of oxidation is observed and required during cell cycle progression), an excessive ROS production may harm proteins, lipids and nucleic acids. For this reason, cells normally maintain a “physiological” level of ROS, by using scavengers, that convert reactive oxygen species into unharmful, non-reactive. These scavengers include the enzyme superoxide dismutase (SOD), catalase, glutathione peroxidase, all transforming ROS in non-reactive oxygen or water.

Protein Kinase A (PKA): protein that modulates other proteins’ activation/inactivation/functioning by adding a chemical group (a phosphate group). The PKA-induced modification is called phosphorylation.

 

References:

Giepmans et al., Science, 2006

Shimomura et al., 1962

Sano et al., Journal of Cell Science, 2014

Jiang et al., PNAS, 2004

Nguyen and Tsien, Nature Review Cancer, 2013,

Tsien, Annual Review Biochemistry, 1998