It's the fluorescent protein, stupid!

Author: Yuezhe Li

Ever since green fluorescence protein was discovered in jellyfish, fluorescent proteins have been widely used in biological studies to tag their protein-of-interest with different colors of fluorescent proteins. What is less often talked about is that fluorescent protein tags can completely mess up your experiments by changing the behavior of your protein-of-interest. 

Protein Structure GFP

​What is a fluorescent protein?
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The first fluorescent protein, green fluorescence protein, also known simply as GFP, is isolated from jellyfish, Aequorea victoria. After the discovery of the first GFP (wtGFP), scientists worked to improve GFP for experimental use. Some early improvements focused on making GFP brighter and less likely to dimerize through genetic mutations. This work led to the development of an optimized GFP (eGFP), one of the GFP proteins that are still widely used in biological labs. Scientists Roger Tsien, Osamu Shimomura, and Martin Chalfie won the 2008 Nobel Prize in Chemistry for their discovery and development of GFP. ​

Fluorescent proteins of different colors were either engineered through genetic mutations or discovered from other species. For example, mutations in GFP led to the development of YFP, a yellow fluorescent protein that is highly similar to GFP.  Mutations also led to the development of pHluorin, a pH-sensitive GFP variant. Scientists also engineered mNeonGreen, a very bright yellow-green fluorescent protein derived from B. lanceolatum. The diversity of fluorescent proteins is exemplified in a picture drawn with bacteria that express fluorescent proteins of different colors.

Many fluorescent proteins are engineered with a special goal in mind; pHluorin is an example. It was engineered to mark out secretion events in cells. pH changes when secretion occurs. Inside secretory granules, the environment is acidic. In the extracellular space, the environment is neutral. pHluorin emits low fluorescence in an acidic environment and is brighter in a neutral environment. This means that when a protein with a pHluorin tag is secreted, a puff is observed (see video).

When your secretion marker prevents secretion 

The postdoc in my lab is interested in studying protein secretion. He uses a mouse pancreatic β cell line that secretes insulin to study this process. To capture secretion events under the microscopy, he used pHluorin-tagged insulin that is supposed to give puffs when secretion happens, as discussed before. 
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Yet somehow, despite literature suggesting YFP-tagged insulin is secreted in β cell lines in response to glucose stimulation, the postdoc did not observe similar phenotypes when he used pHluorin-tagged insulin. In theory, pHluorin is a much better secretion marker than YFP, because it is more sensitive to pH. However, he saw little puffs after cells were stimulated. Out of desperation, my PI asked him to test whether GFP-tagged insulin is secreted. The fluorescence intensity change of GFP in response to pH change is similar to pHluorin: lower in acidic pH, brighter in neutral pH, but is less sensitive than pHluorin. To everyone’s surprise, he observed ample secretion events when he used GFP-tagged insulin! 

It is never clear why pHluorin-tagged insulin cannot be secreted efficiently. After all, pHluorin has been used in secretion related studies for years. However, it is always a cautionary lesson that your seem-to-be-perfect fluorescent protein tag can be the one causing trouble. 
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​When your new brighter fluorescent protein tag changes receptor localization

I am interested in studying insulin receptor localization and signaling. It was previously observed that insulin receptors localize to a specific organelle, the primary cilium, under some conditions. I was interested in how this localization changes insulin receptor signaling and the kinetics. 

I observed this localization using immunofluorescence and probed for the GFP-tagged insulin receptor. However, I could never capture the receptor within the proper localization when I used the mNeonGreen-tagged insulin receptor. My Western blot gave me a tantalizing clue. To my surprise, the Western blot showed the two differently-tagged receptors have very different molecular weights. This should not be the case, because these two fluorescent proteins were similar in weight, and the insulin receptors were tagged the same way.  Further immunostaining showed that while GFP-tagged insulin receptors localized to the plasma membrane, the place the insulin receptor should have been,  mNeonGreen-tagged insulin receptor did not localize to the plasma membrane, instead, it was inside the cytoplasm! 

To conclude, we think the different fluorescent protein tags went through different post-translational processing, causing the GFP-tagged receptor to be correctly localized, but the brighter fluorescent protein tag caused receptor mislocalization.

Conclusion

Sometimes it seems easy to apply published reagents or methods to your own experiments. What is less discussed is the danger of it. In my past three years of training as a cell biologist, I have come to appreciate the complexity of biology and the delicacy of proteins and cells. It seems that we never have the full picture of how cells function. We can help ourselves by taking cautionary steps, understanding no reagent is guaranteed, and making sure there is minimal perturbation to the subject-of-interest. ​​​

Author

Yuezhe Li, @YuezheL
PhD candidate studying biomedical sciences at UCONN Health researching ciliopathy and diabetes. Also loves baking, reading, and traveling.​