Cellular Control – at the Flick of a Light Switch

What if you could turn on an enzyme inside a living cell—or release a cellular factor from its anchor—with the flick of a light switch?

Researchers at the University of Alberta’s Department of Chemistry have developed a new tool for manipulating biochemical processes within cells using light. By applying the unique properties of a photoconvertible fluorescent protein called mMaple, the team created such a light switch, a photocleavable protein called PhoCl (pronounced “focal”).

mMaple, whose name was inspired by the green-to-red color change of maple leaves as seasons transition, undergoes a light-dependent conformational change. Dr. Robert E. Campbell’s team engineered PhoCl to cleave into two pieces when exposed to light.

This novel optogenetic tool is especially useful for applications that involve manipulating cellular processes. For example, PhoCl can be used to create “caged” proteins that will not become activated until exposed to light. Researchers link one terminus of PhoCl to a cellular enzyme and the other terminus to an inhibitor, “caging” the enzyme and preventing it from performing its function. Upon exposure to violet light, PhoCl is cleaved to separate the inhibitor from the enzyme, thus activating the enzyme at the user’s command.

The cleavage mechanism of PhoCl is particularly useful for the activation of proteins within a specific location of a cell. Because intact PhoCl is fluorescent, researchers can visualize its location and movement within the cell and have control over when it cleaves. Upon cleavage, the fluorescence is quenched, enabling users to visually determine where the event took place.

As Allele Biotechnology & Pharmaceuticals is a licensed distributor of plasmids containing the gene for mMaple, the development of PhoCl is particularly exciting news to us and our customers. Interested readers can learn more about PhoCl in their paper published in Nature Methods.

Making the Most of Microscopy with mNeonGreen

Three years since our flagship fluorescent protein was published in Nature Methods, mNeonGreen is still shining bright. mNeonGreen is a green-yellow fluorescent protein that has been shown to be highly useful in optical assays from general imaging to FRET to super resolution microscopy applications and more.

What makes this fluorescent protein such a hit among researchers?

Since the cloning of green fluorescent protein (GFP) over 20 years ago, fluorescent proteins have become a standard research tool, enabling labeling and imaging of individual proteins within a cell in real time. To make these probes brighter, faster folding, and to cover a wider range of the visible spectrum, new fluorescent proteins are continually being developed. mNeonGreen was engineered by scientists at Allele Biotech from a protein isolated from Branchiostoma lanceolatum, a marine invertebrate, and is the brightest monomeric green-yellow fluorescent protein ever characterized.

Perhaps the most common use of fluorescent proteins today is genetically fusing them to a protein of interest to image protein localization. Unfortunately, many researchers are unaware that many fluorescent proteins – such as GFP – are prone to forming noncovalent dimers, which can lead to significant artifacts. True monomeric fluorescent proteins such as mNeonGreen are less likely to affect the localization, dynamics, or normal behavior when fused to proteins of interest.

For quantitative imaging and live-cell imaging applications, arguably the most critical parameters to consider when choosing fluorescent proteins are brightness and photostability. mNeonGreen has high fluorescence brightness, so less light can be delivered to the cells to collect ample signal intensity, resulting in less phototoxicity. mNeonGreen also has superior photostability, meaning it can undergo many excitation-emission cycles before photobleaching occurs. Because of these properties, mNeonGreen has been shown to be very effective as a fluorophore in fluorescence resonance energy transfer (FRET) – both as an acceptor and a donor.

The community of biologists taking advantage of super-resolution fluorescence microscopy (Nobel Prize in Chemistry 2014) is rapidly growing. But the ability to resolve cellular structural features depends on the chosen fluorophore’s brightness, labeling density, and the stability of the dark state. mNeonGreen is not only extraordinarily bright, but also can be driven into a temporary dark state by light irradiation, making it a useful tag for single-molecule super-resolution imaging of proteins.

Researchers around the world continue to develop novel applications for mNeonGreen. Recently, mNeonGreen was used to create a genetically encoded voltage sensor that can be used to image subcellular changes in electrical activity. Researchers fused mNeonGreen to a light-sensitive ion channel in neurons, linking mNeonGreen fluorescence with the membrane voltage to create an optical readout for neuronal activity with unprecedented speed and accuracy.

Choosing an appropriate fluorescent protein for an assay is often a source of confusion for researchers. In many cases, the selection of a fluorescent protein is motivated by convenience (e.g., availability of the construct) rather than its performance for a given assay. If mNeonGreen seems like the right fluorescent protein for your assay, we at Allele Biotech have made it easy and painless for researchers to get their hands on it. Laboratories can license mNeonGreen for full use at a low cost.

Questions about licensing or whether mNeonGreen is really right for your lab? Contact fp@allelebiotech.com.

References:
“A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.”
Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B.R., Allen, J.R., Day, R.N., Davidson, M.W., Wang, J.
2013 Nat Methods, 10(5), 407-409. doi:10.1038/nmeth.2413

“High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor.”
Gong, Y., Huang, C., Li, J. Z., Grewe, B. F., Zhang, Y., Eismann, S., & Schnitzer, M. J.
2015 Science, 350(6266), 1361-1366. doi:10.1126/science.aab0810

Researchers use GFP nano antibody to study organ growth

Single-domain nano antibodies have a broad range of applications in biochemistry due to their small size, high affinity, and high specificity. Now, a team of researchers from the University of Basel and the University of Zurich has demonstrated that nano antibodies can be used for research in complex living organisms such as Drosophila, uncovering another new and exciting application for nano antibodies.

The team used nano antibodies to develop an assay for studying morphogens, molecules that regulate the pattern of tissue growth and the positions of various cell types within tissue. Morphogens form long-range concentration gradients from a localized source, ultimately determining the fate and arrangement of cells that respond to that gradient. Drosophila is a classic model system for understanding how morphogens regulate organ development. One morphogen called Dpp controls uniform proliferation and growth of the wing imaginal disc. Yet because Dpp is an extracellular, diffusible protein, it is difficult to immobilize in situ. Therefore, despite over 20 years of studying the role of Dpp as a morphogen, the lack of a dynamic system for controlling Dpp gradients has prevented researchers from understanding precisely how Dpp governs development of the wing disc.

By developing a novel synthetic system using nano antibodies, the researchers were able to modulate the concentration gradient of Dpp at the protein level. Their system—coined “morphotrap”—uses a membrane-bound GFP nano antibody to “trap” GFP-tagged Dpp at different locations along the wing imaginal disc. By tethering Dpp in a controlled spatial manner, researchers were able to determine how Dpp gradients affect wing disc development. They discovered that the gradient of Dpp is required for the patterning of the wing disc but not for lateral growth, disproving one of the field’s popular theories that address the role of Dpp. In addition to resolving the controversy with respect to the role of Dpp as a morphogen, this study pioneers a new method for using nano antibodies in situ.

“Dpp spreading is required for medial but not for lateral wing disc growth.”
Harmansa S., Hamaratoglu F., Affolter M., Caussinus E.
Nature. 2015 Nov 19;527(7578):317-22. doi: 10.1038/nature15712. Epub 2015 Nov 9.

Tags: Allele Biotech, anti-GFP, camelid antibodies, Fluorescent proteins, morphogens, nanobodies, VHH

Visualizing Endogenous Synaptic Proteins in Living Neurons

The recently published method is based on the generation of disulfide-free “intrabodies”, a structure from the 10th fibronectin type III domain known as FingRs. These affinity molecules were fused to GFP for direct fluorescence miscroscopy. The FingRs do not need di-sulfite bonds and are therefore better folders in mammalian cells. Specifically, a library was screened with in vitro display to identify FingRs that bind two synaptic proteins, Gephyrin and PSD95. After the initial selection, the researchers from USC secondarily screened binders using a cellular localization assay to identify potential FingRs that bind at high affinity in an intracellular environment. As it turned out, only 10-20% of the original positive clones bind well inside the cells, suggesting this type of further screening was a critical step.

The expression of intrabody is transcriptionally regulated by the target protein through a ZFN-repressor fusion. This transcriptional control system matches the expression of the intrabody to that of the target protein regardless of the target’s expression level. This design virtually eliminates unbound FingR, resulting in very low background that allows unobstructed visualization of the target proteins. As result, the FingRs presented in this study enabled live cell visualization of excitatory and inhibitory synapses, and apparently without affecting neuronal function.

Technically, the reason to use in vitro mRNA display was required by the need to use a large library (>10exp12, beyond the limit of the more commonly used phase display) to find good binders. A similar visualization system can be established using more potent affinity domains such as the VHH single-domain antibodies that have only one, sometimes dispensable, di-sulfite bond. The VHH domain nanobodies can be more easily isolated from camelid animals. Another improvement to the visualization system can be made by using stronger, superresolution-ready FPs such as mNeonGreen or mMaple to enable single molecule imaging, which is particularly interesting for studying synapses and applied to the BRAIN initiative.

Gross et al. Neuron, June 2013, http://www.ncbi.nlm.nih.gov/pubmed/23791193

Tags: alpaca antibodies, BRAIN, FingR, fluorescent protein tag, intrabodies, mNeonGreen, nAb, nanobodies, neuron, single domain, Synapses, VHH, VHH antibodies

The Development of mNeonGreen

This week our most recent publication, “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum” will be published in Nature Methods. It has already been viewable online for some time now, here is a link. We believe this new protein possesses a great deal of potential to advance the imaging fields through enhanced fluorescent microscopy. mNeonGreen enables numerous super resolution imaging techniques and allows for greater clarity and insight into one’s research. As a result of this we are taking a new approach at Allele for distribution of this protein, and here we will describe the history of the protein and some of the factors that led us down this path.

mNeonGreen was developed by Dr. Nathan Shaner at Allele Biotechnology and the Scintillon Institute through the directed evolution of a yellow fluorescent protein we offer called LanYFP. LanYFP is a super bright yellow fluorescent protein derived from the Lancelet fish species, characterized by its very high quantum yield, however, in its native state LanYFP is tetrameric. Dr. Shaner was able to monomerize the protein and enhance a number of beneficial properties such as photostability and maturation time. The result is a protein that performs very well in a number of applications, but is also backwards compatible with and equipment for GFP imaging.

Upon publication there was a question of how distribution should be structured. How would we make this protein available to researchers in a simple manner was a very difficult challenge? We also relied heavily on Dr. Shaner’s knowledge and experience in these matters, as he related his experiences to us from his time in Roger Tsien’s lab at UCSD. When the mFruits was published their lab was inundated with requests. The average waiting period was 3 months to receive a protein and they required a dedicated research technician to handle this process. Eventually the mFruits from the Tsien lab were almost exclusively offered through Clontech. Thus we decided that Allele Biotechnology would handle the protein distribution and take a commercial approach to drastically decrease the turnaround time. The next challenge we faced was how to charge for this protein. Due to the cost of developing this protein, which was fully funded by Allele, there is a necessity to recoup our investment and ideally justify further development of research tools, but we also understand the budget constraints every lab now faces. From this line of thinking we conceived our group licensing model; we wanted to limit the charge to $100 per lab. The way this is fiscally justifiable is having every lab in a department or site license the protein at this charge, including access to all related plasmids made by us as well as those generated by other licensed users (Click here for our licensing page). The benefit we see to this is that the protein is licensed for full use at a low cost, and collaboration amongst one’s colleagues is not only permissible, it’s encouraged. We saw this as a win-win situation. We would recoup our cost and invest in further fluorescent protein research, and our protein costs would not be a barrier to research and innovation.

The granting of a license to use but not distribute material is not unique to commercial sources. Although academic material transfer agreements typically contain specific language forbidding distribution of received material beyond the recipient laboratory, some researchers choose to disregard these provisions. Unfortunately through this action they are disrespecting the intellectual property rights of the original researchers as well as violating the terms of the legal contract they signed in order to receive the material. We believe most researchers choose to respect the great deal of effort that goes into the creation of research tools for biology and do not distribute any material received from other labs without their express permission. However for a company that funds its own basic research our focus is often on the former example rather than the latter. We believe that this focus artificially drives up the costs of licensing a fluorescent protein and obtaining the plasmid, thus we have chosen to believe researchers will respect our intellectual property as long as we are reasonable in our distribution which is something we have truly striven for.

Additionally we believe the broad-range usage of a superior, new generation FP is an opportunity to advocate newer technologies that can be enabled by mNeonGreen, together with a number of Allele’s other fluorescent proteins (such as the photoconvertible mClavGR2, and mMaple). These new imaging technologies are called super resolution imaging (MRI). They provide researchers with a much finer resolution of cellular structures, protein molecule localizations, and protein-protein interaction information. We have started the construction of a dedicated webpage to provide early adopters with practical and simple guidance, click here to visit our super resolution imaging portal.

Tags: cell imaging, fluorescent protein, FP, GFP, mNeonGreen, Superresolution