Acid-tolerant green fluorescent protein for bioimaging

January 4, 2018, Osaka University
A schematic of development of Gamillus. Gene of the fluorescent protein was cloned from tentacles of flower hat jellyfish, and was engineered to improve the monomeric property and the brightness. (The flower hat jellyfish was provided by Mr. Kamoizumi in Kamo Aquarium, Yamagata, Japan.) Credit: Aquarium and Osaka University

Osaka University researchers develop new green fluorescent protein that can withstand low pH environment for imaging of acidic organelles

Visualizing cellular components and processes at the molecular level is important for understanding the basis of any biological activity. Fluorescent proteins (FPs) are one of the most useful tools for investigating intracellular molecular dynamics.

However, FPs have usage limitations for imaging in low pH environments, such as in acidic organelles, including endosomes, lysosomes, and plant vacuoles. In environments of pH less than 6, most FPs lose their brightness and stability due to their neutral pKa. pKa is the measure of acid strength; the smaller the pKa is, the more acidic the substance is.

"Although there are reports of several acid-tolerant green FPs (GFPs), most have serious drawbacks. Furthermore, there is a lack of acid-tolerant GFPs that are practically applicable to bioimaging," says Hajime Shinoda, lead author of an Osaka University study that aimed to design acid-tolerant monomeric GFP that is practically applicable to live-cell imaging in acidic organelles. "In the current study, we developed an acid-tolerant GFP. We called it Gamillus."

Acid-tolerant green fluorescent protein for bioimaging
Figure 2. (Left) pH-dependent fluorescence property of Gamillus and EGFP. (Right) Fluorescence images of HeLa cells expressing Gamillus or EGFP. Gamillus enables fluorescence observation of protein migration to lysosomes caused by macroautophagy. Scale bar, 10 μm. Credit: Osaka University

Gamillus is a GFP cloned from Olindias formosa (flower hat jellyfish) and exhibits superior acid tolerance (pKa=3.4) and nearly twice as much brightness compared with the reported GFPs. The fluorescence spectrum is constant between pH4.5 and 9.0, which falls between the intracellular range in most cell types. X-ray crystallography (a technique used for determining the atomic and molecular structure of a crystal, in this case, a Gamillus crystal) and point mutagenesis suggest the acid tolerance of Gamillus is attributed to stabilization of deprotonation in its chemical structure. The findings were published in Cell Chemical Biology.

"The applicability of Gamillus as a molecular tag was shown by the correct localization pattern of Gamillus fusions in a variety of cellular structures, including ones that are difficult to target," corresponding author Takeharu Nagai says. "We believe Gamillus can be a powerful molecular tool for investigating unknown biological phenomena involving acidic organelles, such as autophagy."

Explore further: Study finds harmful protein on acid triggers a life-threatening disease

More information: Hajime Shinoda et al. Acid-Tolerant Monomeric GFP from Olindias formosa, Cell Chemical Biology (2017). DOI: 10.1016/j.chembiol.2017.12.005

Related Stories

Cloned sorghum is aluminum tolerant

February 23, 2010

(PhysOrg.com) -- Leon Kochian and colleagues have cloned a unique sorghum gene that is being used to develop sorghum lines that can withstand toxic levels of aluminum in the soil, a consequence of acidic soils.

Behind a marine creature's bright green fluorescent glow

July 1, 2014

Pushing closer to understanding the mechanisms behind the mysterious glow of light produced naturally by certain animals, scientists at Scripps Institution of Oceanography at UC San Diego have deciphered the structural components ...

Recommended for you

A new polymer raises the bar for lithium-sulfur batteries

January 18, 2018

Lithium-sulfur batteries are promising candidates for replacing common lithium-ion batteries in electric vehicles since they are cheaper, weigh less, and can store nearly double the energy for the same mass. However, lithium-sulfur ...

Looking to the sun to create hydrogen fuel

January 18, 2018

When Lawrence Livermore scientist Tadashi Ogitsu leased a hydrogen fuel-cell car in 2017, he knew that his daily commute would change forever. There are no greenhouse gases that come out of the tailpipe, just a bit of water ...

The early bits of life

January 18, 2018

How can life originate before DNA and genes? One possibility is that there are natural processes that lead to the organisation of simple physical objects such as small microcapsules that undergo rudimentary forms of interaction, ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.