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.
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: Kamo Aquarium and Osaka University
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."
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."
Saori Obayashi | EurekAlert!
Overlooked molecular machine in cell nucleus may hold key to treating aggressive leukemia
23.04.2019 | Cincinnati Children's Hospital Medical Center
Bacteria use their enemy -- phage -- for 'self-recognition'
23.04.2019 | Chinese Academy of Sciences Headquarters
Researchers led by Francesca Ferlaino from the University of Innsbruck and the Austrian Academy of Sciences report in Physical Review X on the observation of supersolid behavior in dipolar quantum gases of erbium and dysprosium. In the dysprosium gas these properties are unprecedentedly long-lived. This sets the stage for future investigations into the nature of this exotic phase of matter.
Supersolidity is a paradoxical state where the matter is both crystallized and superfluid. Predicted 50 years ago, such a counter-intuitive phase, featuring...
A stellar flare 10 times more powerful than anything seen on our sun has burst from an ultracool star almost the same size as Jupiter
A localization phenomenon boosts the accuracy of solving quantum many-body problems with quantum computers which are otherwise challenging for conventional computers. This brings such digital quantum simulation within reach on quantum devices available today.
Quantum computers promise to solve certain computational problems exponentially faster than any classical machine. “A particularly promising application is the...
The technology could revolutionize how information travels through data centers and artificial intelligence networks
Engineers at the University of California, Berkeley have built a new photonic switch that can control the direction of light passing through optical fibers...
Physicists observe how electron-hole pairs drift apart at ultrafast speed, but still remain strongly bound.
Modern electronics relies on ultrafast charge motion on ever shorter length scales. Physicists from Regensburg and Gothenburg have now succeeded in resolving a...
17.04.2019 | Event News
15.04.2019 | Event News
09.04.2019 | Event News
23.04.2019 | Information Technology
23.04.2019 | Earth Sciences
23.04.2019 | Life Sciences