HOW ACIDIC IS A CELL’S DIGESTIVE SYSTEM? A NEW FLUORESCENT PROTEIN PAIR CAN TELL YOU

Michelle Ehrenberger

Title: “pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments”
Authors: Burgstaller, S., Bischof, H., Gensch, T., Stryeck,
S.,Gottschalk, B., Ramadani-Muja, J., Eroglu, E., Rost, R.,
Balfanz, S., Baumann, A., Waldeck-Weiermair, M., Hay,
J.C., Madl, T., Graier, W.F., Malli, R.
Journal: ACS Sensors
Year: 2019
https://pubs.acs.org/doi/10.1021/acssensors.8b01599
Featured image and figures used with permission via ACS Editors’ Choice open access

Have you ever wondered how a cell digests its food? It turns out that a teeny tiny cell digests its food in much the same way you do: with special acidic compartments that break down nutrients. In human beings, this compartment is the stomach, and if you’ve experienced heartburn (a condition where stomach juices rise painfully up into your esophagus [1]) then you have some idea of what digestive acid feels like and why it needs to be kept separate from the rest of your body. An individual cell may have many digestive compartments, called lysosomes, and they also have other membrane-bound cellular compartments called endosomes that shuttle nutrients from the outer cell membrane to the lysosomes to be broken down. While scientists know that most of the inside of a cell is neutral (around pH 7.3), that endosomes are slightly acidic (around pH 6), and that lysosomes are highly acidic (around pH 4), they still have trouble getting an exact measurement for a pH of a single cellular compartment. Having a tiny probe that can measure the acidity at a specific spot in a cell would be especially important for understanding diseases—including cancer—that involve dysregulation of pH inside cellular compartments.

But how do you measure the pH of something as tiny as a single lysosome? By using a pH-dependent fluorescent protein! Fluorescent molecules absorb a specific high-energy wavelength of light (for example, blue light with a 475nm wavelength) but emit a specific lower-energy wavelength of light (such as 525nm yellow light). Because fluorescence is dependent on a molecule’s structure, chemical reaction with an acidic environment can cause some molecules to temporarily lose their fluorescence. This is the case for the Enhanced Yellow Fluorescent Protein (EYFP) (Fig. 1a).

EYFP absorbs blue light and fluoresces yellow light when it’s in a neutral or alkaline (the opposite of acidic) environment, but not in an acidic environment. That makes it a great choice as a probe to measure the acidity of different cellular compartments. Since EYFP is a protein, scientists can modify the DNA of a cell to include the EYFP gene, getting the cell itself to make the protein probe. They can also modify the gene to include another protein that always goes to a specific cellular compartment, such as a protein called LC3B that goes to the endosomes and lysosomes. When EYFP is attached to LC3B, scientists can image the fluorescence—and therefore the acidity—of specific subcellular compartments.

Figure 1. FRET pair fluorescence. (a) Scheme of pH-Lemon FRET, blue protein is mTurquoise2, yellow is EYFP. (b) Fluorescence intensity of proteins individually at different pH. (c) Ratio of EYFP fluorescence (FRET) to mTurquoise2 fluorescence (CFP) at different pH.

However, one more step is still needed to make the final probe. While EYFP fluorescence intensity would be a pretty good indicator of pH, scientists can be more certain that their probe is working and can also start to quantify the acidity when they use a pair of fluorescent proteins, specifically a FRET (Förster resonance energy transfer) pair, instead. In FRET, the wavelength of light emitted by one fluorescent protein (like mTurquoise2) is absorbed by another fluorescent protein (like EYFP). As can be seen in Fig. 1a, in a neutral or alkaline cellular compartment, mTurquoise2 absorbs 430nm blue light and emits 475nm blue light which in turn causes EYFP to emit 525nm yellow light. In an acidic environment, only 475nm blue light is emitted, since EYFP cannot fluoresce.

Using a FRET pair is beneficial, since a high intensity of yellow light seen when using EYFP alone could simply be due to a high concentration of EYFP in one spot. With the FRET pair however, as can be seen in Fig. 1b and 1c, the ratio of mTurquoise2 fluorescence to EYFP fluorescence can be used to measure the acidity regardless of protein concentration in that spot. From pH 4 through pH 8, the fluorescence of mTurquoise2 remains relatively the same (Fig. 1b) while the fluorescence of EYFP is very pH-dependent. Having designed this very promising probe, the scientists named the FRET pair “pH-Lemon” and sent it on its maiden voyage through the cell, sometimes untethered and sometimes bound to the LC3B protein to direct its subcellular journey.

What they found was very exciting! pH-Lemon in the endoplasmic reticulum (ER) and mitochondria (Mito) showed a low ratio of mTurquoise2 : EYFP fluorescence (Fig. 2) which indicates a neutral or alkaline environment, as is expected for these cellular compartments. The endosomes and lysosomes (containing pH-Lemon-LC3B), however, are all expected to be acidic, and pH-Lemon shows that they are! Thus, this FRET pair has proven itself as a quantifiable subcellular pH probe.

Figure 2. Images of human HeLa cells with pH-Lemon in the (a) endoplasmic reticulum, (b) mitochondrial matrix, (c) endosomes and lysosomes. Scale bar is 10µm. Ratios given are fluorescent ratio of mTurquoise2 to EYFP during FRET.

pH-Lemon has some other advantages as well. It seems to be non-toxic to mammalian cells, which non-protein fluorescent molecules often aren’t. It can also be re-used, unlike another common probe called LysoTracker Red DND-99 which can detect acidic environments but is no longer usable after being in a neutral environment. It is also a great complement to other pH-sensing proteins such as one called SypHer that is optimal for use in more alkaline environments than pH-Lemon.

Overall, pH-Lemon is likely at the beginning of a very promising career investigating the acidity of subcellular compartments, uncovering the secrets of pH regulation in cells that are healthy, diseased, or even cancerous.

[1] https://www.niddk.nih.gov/health-information/digestive-diseases/acid-reflux-ger-gerd-adults

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