In a previous blog post we discussed how fluorescent proteins can be used to construct biosensors, biological tools that monitor processes or detect molecules. Here we’ll be diving into the details surrounding SF-iGluSnFr, a recently upgraded biosensor designed to detect glutamate.
The Origins of iGluSnFr
iGluSnFr was developed in 2013 by Loren Looger’s lab to give researchers the ability to monitor glutamate release from neurons and other brain cells in vivo. Glutamate plays a variety of roles in synaptic communication and can trigger other forms of neuronal signaling and regulation.
To create iGluSnFr, the Looger lab wedged GFP between two halves of a bacterially derived periplasmic glutamate binding protein known as GltI. When the resulting fusion protein binds to glutamate, conformational changes lead to an increase in fluorescence intensity from the inserted GFP.
After characterizing iGluSnFr in vitro, showing that it is selectively activated by glutamate, and tweaking it to improve its responsiveness, the Looger lab showed that iGluSnFr could detect neuronal activity in C. elegans, zebrafish, and mice.
Upgrades to iGluSnFr
Although a useful tool, this first version of iGluSnFr wasn’t optimal for experiments requiring high signal to noise ratios or imaging of high frequency glutamate signaling events. The Looger lab therefore improved upon iGluSnFr in 3 major ways detailed in a preprint by Marvin et al. The researchers did the following:
- Increased iGluSnFr’s brightness
- Created new iGluSnFr variants with different glutamate binding kinetics
- Created blue and yellow color iGluSnFr variants
In their first change to iGluSnFr, Marvin et al swapped eGFP for sfGFP to create SF-iGluSnFr. This improved variant has higher expression levels in bacteria and and ultimately produces stronger fluorescent signals upon sensing glutamate in mouse and ferret models.
Increased Versatility through Varied Glutamate Binding Kinetics
Although the original iGluSnFr had a physiologically relevant 4 μM affinity for glutamate in cell culture, this relatively low level of affinity affected performance in the following settings:
- Experimental setups that require low frequency signal detection, increased affinity, and high signal to noise ratios
- Identification of discrete, high frequency events - these require lower affinity (through faster off rates) to identify signals from individual events
To overcome these issues, Marvin et al mutated a region of the iGluSnFr fusion protein known to be important for affinity. Mutation of this so-called “hinge region” resulted in the identification of two useful variants:
- SF-iGluSnFr.A184S (increased affinity for glutamate)
- SF-iGluSnFr.S72A (decreased affinity for glutamate)
Marvin et al. further show that the A184S variant produces a more intense fluorescent signal in response to lower concentrations of glutamate as compared to iGluSnFr. The S72A variant on the other hand, is better at detecting repeated high frequency events like neuronal glutamate release upon electrical stimulation. With these variants, researchers can choose the specific tool that best suits their experimental needs.
New SF-iGluSnFr Colors
The original iGluSnFr only came in one color - green. A researcher might need more colors to image separate signalling events in nearby cells or to easily identify glutamate signaling in specific cell types. Marvin et al therefore swapped our the GFP chromophore in iGluSnFr and optimized the GltI-fluorescent protein linkages where necessary to produce blue (SF-Azurite-iGluSnFr) and yellow (SF-Venus-iGluSnFr) variants. Indeed, beyond adding new colors, the yellow variant can also be imaged by cheap 1030 nm two photon femtosecond lasers that can excite multiple areas in a single sample. With these SF-iGluSnFr constructs, scientists have both new iGluSnFr colors and new ways to excite them!
Using SF-iGluSnFr in Your Lab
The new SF-iGluSnFr variants should enable scientists to robustly detect signaling events in the brain and beyond. The Looger lab focuses on SF-iGluSnFr applications in neurobiology in the papers discussed here, but, as they mention in their 2013 work, glutamate signaling occurs in a many contexts and could be useful for scientists working in organisms ranging from bacteria to animals to plants.
The SF-iGluSnFr variants come in AAV, mammalian expression, and bacterial expression vectors available from Addgene. The Looger Lab has provided a brief breakdown of the promoter systems you can use to express the SF-iGluSnFr variants:
- CAG: Generic strong promoter. Formulated as AAV-DJ for neuronal expression.
- CAG-FLEX: CAG promoter, FLEXed for expression in Cre-expressing cells.
- hSynap: Human Synapsin-1 promoter. Good for neuronal expression.
- hSynap-FLEX: Synanpsin-1 promoters, FLEXed for expression in Cre-expressing cells.
- GFAP: Glial fibrillary acidic protein (promoter). Good for Gliall expression (not neurons).
- pMinDis: CMV promoter. Good for transient expression in all cells types and subcloning. Based on pDisplay from Invitrogen, but with the HA tag removed.
- pRSET: T7 promoter for expression of soluble protein in bacteria. Lacks secretion signal and transmembrane domain.
We’re excited to see how you use these great new tools!
1. Marvin, Jonathan S., et al. "Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR." bioRxiv (2017): 235176.
Additional Resources on the Addgene Blog
- Learn about other Fluorescent Biosensors
- A Practical Approach to Choosing the B(right)est Fluorescent Protein
- Visit the Fluorescent Protein Featured Topic Page
Resources at Addgene.org