Oliver Griesbeck of the Max Planck Institute for Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the very beginnings of the field. Those engineered sensors were designed to replace synthetic calcium dyes, which had been in use since the 1980s.
“Synthetic dyes were the standard in the field, but there is one problem: how to get that into the cells of interest,” Griesbeck said. Because they are chemical compounds, they have to be applied or injected, and they don’t always end up where you want them to go.
Griesbeck is motivated by a particular interest in monitoring the activity and biochemistry of living neurons in an effort to understand the connection between molecular- and cellular-level events and behavior. It’s a problem that he considers “one of the greatest challenges of neuroscience.”
Hot Plasmids: Twitch Sensors
Griesbeck’s recent contribution to the field is a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most notably, the Twitch calcium sensors, which are available at Addgene, can be used for ratiometric in vivo imaging. Griesbeck explains, while these Twitch sensors are always bright, they change color as they shift from "off" to "on" in the presence of calcium. This color change can be observed under a microscope using commercially available beam splitters. In addition to imaging neurons, Griesbeck adds that this color change has a particular advantage for observing events taking place inside cells that are on the move, such as T lymphocytes.
Griesbeck and his colleagues began by isolating a calcium-binding protein, troponin C (TnC), from muscle cells. They identified a toadfish TnC variant which had a high-affinity for calcium binding and developed a version of the protein with as few calcium-binding sites as possible for added sensitivity.
As reported in Nature Methods, the FRET changes of these Twitch sensor variants were optimized by testing various linker configurations in a large-scale functional screen in bacteria. Sensor variants were then refined by a secondary screen in rat hippocampal neuron cultures. The optimized Twitch sensors allowed the researchers to see tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes, too, making them a versatile tool for application in both neuroscience and immunology.
Don’t Be Shy: Ratiometric Imaging for Beginners
Griesbeck says his calcium indicators can be used in mouse, Drosophila, C. elegans, and zebrafish – essentially all of the standard organisms for which extensive genetic tools are available. While he recognizes that some beginners may have a tendency to shy away from ratiometric imaging, he recommends they give it a shot.
“Beginners sometimes think it’s complicated, but actually it is very good,” he said. “It gives you more information than intensity-based readouts.”
Griesbeck also points out that the approach he and his colleagues took to optimize sensitivities can now be applied to dozens of other published FRET-based sensors. For example, his team is now applying it to improve upon an indicator they developed and published in 2010 for visualizing the transcription factor cAMP-responsive element-binding protein (CREB) in living cells.
“Now we are making this really good,” Griesbeck said. “We know CREB is really important, but where is it really important and when? That is not known.”
Maarten Merkx of Technische Universiteit Eindhoven has similar interest in FRET-based sensor proteins, particularly for the intracellular imaging of transition metal ions, such as zinc, copper, and iron.
“These are essential metal ions, but they are also toxic,” Merkx explained. New tools were needed to measure them.
Merkx says, while in principle adapting a FRET sensor for monitoring calcium versus zinc should require the simple swapping of binding domains, in practice optimizing the sensors involves plenty of trial and error. Transition metals also present challenges in that they are found in cells at much lower concentrations than calcium, requiring greater sensitivity.
He and his colleagues came up with a solution: they devised self-associating fluorescent domains whose association is disrupted in the presence of a ligand (they stick together in one state, but not the other).
“It’s quite a robust way to make a FRET sensor with a large dynamic range,” he says. Merkx recently applied this “trick” to the development of additional color variants. This resulted in the construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants of mOrange2 and mCherry as donor and acceptor domains. He says, these new colors might now enable observation of the interaction between different molecules like zinc and calcium.
Ultimately, Merkx said he hopes others will find his tools useful, including his zinc sensor and his genetically encoded magnesium sensor, the first of its kind.
“I hope by depositing at Addgene, people will start using them,” Merkx said. “For us, it is one of the main measures of success – whether we have really developed something useful [enough] that other people start using it. For us, it’s like a test.”
- Thestrup et al. Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773. (PubMed)
- Friedrich et al. Imaging CREB activation in living cells. J Biol Chem. 2010 Jul 23;285(30):23285-95. doi: 10.1074/jbc.M110.124545. (Pubmed)
- Lindenburg et al. Robust red FRET sensors using self-associating fluorescent domains. ACS Chem Biol. 2013 Oct 18;8(10):2133-9. doi: 10.1021/cb400427b. Epub 2013 Aug 30. (PubMed)
- Lindenburg et al. MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor. PLoS One. 2013 Dec 2;8(12):e82009. doi: 10.1371/journal.pone.0082009. (PubMed)
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- If you're interested in plasmids for in vivo imaging, check out this blog post.