By twitter request, click-thru for the original resolution of a fellow Janelian’s Nikon Small World 4th place prize winner. Ryan Williamson composed the image.
Drosophila melanogaster visual system halfway through pupal development, showing retina (gold), photoreceptor axons (blue), and brain (green) (1500x)
Nature methods published two papers which extend brainbow-like techniques of stochastic multicolored neuronal labeling into fruit flies. Nature’s summary explains the two methods.
The first technique, called dBrainbow, was developed by Julie Simpson, a neuroscientist at the Howard Hughes Medical Institute’s Janelia Farm Research Campus in Ashburn, Virginia, and her colleagues2. This method uses enzymes called recombinases to randomly delete some of the colour-producing genes from the string, leaving different genes next to the promoter regions in different cells. Individual cells are therefore uniquely coloured and so can be easily distinguished…
The second technique, called Flybow, was developed by Salecker and her colleagues3. They used an enzyme that ‘flips’ pairs of colour-producing genes on the string, leaving different genes next to the promoter region. The ‘flipping’ enzyme is also a recombinase, and so after being inverted, some of the colour-producing genes are randomly deleted. This ensures that all the different genes on the string can potentially end up next to the promoter, and be displayed by individual modified neurons.. Flybow uses a single string of four colours — red, green, blue and yellow.
These techniques will find use in building the structural and functional connectome of the fly.
Three excellent pieces of neuroscience software have been recently updated or freshly released. I have used two of them, Ephus and ScanImage, on a daily basis as primary data collection tools. The third, Neuroptikon, is quite useful for post-hoc illustration of neural circuits.
Ephus is a modular Matlab-based electrophysiology program that can control and record many channels of tools and data simultaneously. Under control of a sophisticated internal looper or external trigger, you can initiate an ephys recording, trigger camera frames, adjust galvo positions, open/close shutters, trigger optical stimulation, punishments, rewards, etc. It is a workhorse program for non-imaging related in vitro and in vivo electrophysiology experiments. Ephus is named for the fabled baseball pitch, and pronounced as “EFF-ess”. As with the pitch, it may trick you at first, but eventually you’re sure to hit a home run. Of course, the name also evokes electrophysiology, which is the fundamental orientation of the project, be it optical or electrical.
Ephus 2.1.0 is a major release, and the only official version at this time. The software is fully described in a publication in Frontiers in Neuroscience. New features include unlimited recording time, with disk streaming, for applications such as EEGs and long traces during in-vivo behavior. A number of additional scripts for in-the-loop control have been added. New configuration/start-up files have been created, with a template to help get up and running quickly. This release also includes a number of bug fixes.
ScanImage is another Matlab-related software program that is used for optical imaging and stimulation of neurons in vitro and in vivo. It finds much use a control platform for 2-photon imaging, glutamate uncaging and laser-scanning photostimulation. An early incarnation is described in this paper by Pologruto, et al. It provides a lot of power right out of the box (bidirectional scanning @ 0.5ms/line, etc) and is easily extensible via custom user function plugins.
Neuroptikon is a sophisticated network visualization tool. It can build Van Essen-like diagrams of any circuit you like, but it is so much more. The direction of communication is animated, and subsets of regions and connections can be brought into focus, which greatly eases the clarity of the network. The diagrams can be built in three-dimensions, to preserve relative topography, or functional grouping. There is simple GUI-based control, while more complex tasks can use a scripting interface. This is great software for anyone who needs to imagine information flow in a complex network.
All three tools are released for free use under the HHMI/Janelia Farm open source license.
Download Here :
“If you find yourself needing to reread this paragraph, perhaps it’s not that well written. Or it may be that you are low on acetylcholine.” Acetylcholine (ACh) is a major modulator of brain activity in vivo and its release strongly influences attention. If we could visualize when and where ACh is released, we could more fully understand the large trial to trial variance found in many in vivo recordings of spike activity, and perhaps correlate that to attentional and behavioral states mediated by ACh transmission.
Back in grad school, when I was desperately trying to figure out what biological question to answer with my GluSnFR glutamate sensor, I ended up in a meeting with Kleinfeld, his grad student Lee Schroder and Palmer Taylor. We plotted a strategy to make a FRET sensor for acetylcholine. Palmer had recently solved crystal structures of an acetylcholine binding protein bound to agonists and antagonists. Snails secrete this binding protein into their ACh synapses to modulate their potency. The structures showed a conformational change upon agonist binding. The hope was that by fusing CFP and YFP to the most translocated bits of the protein, they would be able to see an ACh dependent FRET change. I was skeptical that it would work, as the translocation was much less than with calmodulin-M13 or periplasmic binding proteins used in Cameleon and GluSnFR, but thought was at least worth a shot. FRET efficiency is highly dependent on dipole orientation, not just dipole distance, and you never know how a small conformational change might rearrange the FP dipoles…
Of course, the simple idea didn’t work. Instead of giving up on the first dozen attempts, they kept plugging away at alternative strategies for measuring ACh release, and eventually succeeded. In this Nature Neuroscience report, An in vivo biosensor for neurotransmitter release and in situ receptor activity, Nguyen et al demonstrate a mammalian cell based system for optically measuring ACh levels in an intact brain. They coexpressed M1 muscarinic receptors with the genetically-encoded calcium indicator TN-XXL in HEK293 cells. ACh binding to the M1 receptor induced IP3-mediated calcium influx. This calcium rise was then picked up by the TN-XXL and reported as a change in CFP/YFP fluorescence. The crazy part is that they took this cell culture assay and implanted the cells into the brains of living rats!
The CNiFER in vivo experimental paradigm
In culture, the response was highly sensitive and monotonic (for phasic response section, EC50 of 11 nM, a Hill coefficient of 1.9 and a maximum of ΔR/R = 1.1). In vivo, using two-photon imaging through a cortical window, they were able to see clear ACh responses in frontal cortex from electrical stimulation of the nucleus basalis magnocellularis, typically 200-μs current pulses of 200 μA @ 100Hz for 20-500ms.
This was essentially a in vivo proof of principal experiment, showing that one could image ACh release in spatially and temporally precise regions of the brain. However, the imaging was done under urethane anesthesia, which is a much different brain state than an awake, behaving animal. Are CNiFERs sensitive, powerful and stable enough to determine behavioral states via imaging in an awake animal? Would expressing GCaMP3 (an indicator with greater fluorescence dynamic range) improve the performance of the CNiFER system? We used a very similar assay with ACh applied to HEK cells during the initial screens for better GCaMPs. Or, is the performance more limited by the properties of the M1 receptor and the adapting nature of IP3-mediated calcium dynamics? CNiFERS provide an interesting platform for looking at ACh and potentially other G-protein mediated signaling, but it remains to be seen if labs that aren’t as technically proficient with two-photon rig will find it more useful than cyclic voltammetry for measuring acetylcholine levels.
Nature Neuroscience, 13 (1), 127-132 DOI: 10.1038/nn.2469
Nguyen, Q., Schroeder, L., Mank, M., Muller, A., Taylor, P., Griesbeck, O., & Kleinfeld, D. (2009). An in vivo biosensor for neurotransmitter release and in situ receptor activity
I somehow missed that Josh Vogelstein’s method on action potential detection was published last summer. In Spike Inference from Calcium Imaging Using Sequential Monte Carlo Methods, the authors use a Monte Carlo approach to determine spike times from calcium imaging with superior performance to other deconvolution methods. It does a great job on simulated and in vitro data, I’d love to see performance on real in vivo recordings. If you are serious about calcium imaging, you should definitely get in touch with Josh and see what magic he can do with all that math. You should also ask him about the benefits of linen pants vs. denim, he’s got strong opinions on that subject as well…
Using only strongly saturating and very noisy in vitro fluorescence measurements to infer precise spike times in a ‘‘naturalistic’’ spike train recorded in vitro
Annual Reviews of Neuroscience published their 2009 issue recently. These articles are usually a great way to catch up with a field, particularly when they are recently published. Here are a few that might be of interest to the Brain Windows reader.
Sensory experience and learning alter sensory representations in cerebral cortex. The synaptic mechanisms underlying sensory cortical plasticity have long been sought. Recent work indicates that long-term cortical plasticity is a complex, multicomponent process involving multiple synaptic and cellular mechanisms. Sensory use, disuse, and training drive long-term potentiation and depression (LTP and LTD), homeostatic synaptic plasticity and plasticity of intrinsic excitability, and structural changes including formation, removal, and morphological remodeling of cortical synapses and dendritic spines. Both excitatory and inhibitory circuits are strongly regulated by experience. This review summarizes these findings and proposes that these mechanisms map onto specific functional components of plasticity, which occur in common across the primary somatosensory, visual, and auditory cortices.
Diffusion imaging can be used to estimate the routes taken by fiber pathways connecting different regions of the living brain. This approach has already supplied novel insights into in vivo human brain anatomy. For example, by detecting where connection patterns change, one can define anatomical borders between cortical regions or subcortical nuclei in the living human brain for the first time. Because diffusion tractography is a relatively new technique, however, it is important to assess its validity critically. We discuss the degree to which diffusion tractography meets the requirements of a technique to assess structural connectivity and how its results compare to those from the gold-standard tract tracing methods in nonhuman animals. We conclude that although tractography offers novel opportunities it also raises significant challenges to be addressed by further validation studies to define precisely the limitations and scope of this exciting new technique.
The ultimate goal of neural interface research is to create links between the nervous system and the outside world either by stimulating or by recording from neural tissue to treat or assist people with sensory, motor, or other disabilities of neural function. Although electrical stimulation systems have already reached widespread clinical application, neural interfaces that record neural signals to decipher movement intentions are only now beginning to develop into clinically viable systems to help paralyzed people. We begin by reviewing state-of-the-art research and early-stage clinical recording systems and focus on systems that record single-unit action potentials. We then address the potential for neural interface research to enhance basic scientific understanding of brain function by offering unique insights in neural coding and representation, plasticity, brain-behavior relations, and the neurobiology of disease. Finally, we discuss technical and scientific challenges faced by these systems before they are widely adopted by severely motor-disabled patients.
Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.
Here’s a group of four recent papers that are worth checking out but I don’t have the time to cover. The first provides a set of tools for neuronal circuit tracing. The second pushes super-resolution imaging into fast, live-cell imaging. The third, by a friend from graduate school, uses G-CaMP to make strong claims about olfactory coding in fruit flies. The last reports remarkable data pointing to the distributed nature of conscious perception in humans, which would have been a great data set to reference in my recent talk on free will.
Genetically timed, activity-sensor and rainbow transsynaptic viral tools
We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.
Super-resolution video microscopy of live cells by structured illumination
Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.
Select Drosophila glomeruli mediate innate olfactory attraction and aversion.
Fruitflies show robust attraction to food odours, which usually excite several glomeruli. To understand how the representation of such odours leads to behaviour, we used genetic tools to dissect the contribution of each activated glomerulus. Apple cider vinegar triggers robust innate attraction at a relatively low concentration, which activates six glomeruli. By silencing individual glomeruli, here we show that the absence of activity in two glomeruli, DM1 and VA2, markedly reduces attraction. Conversely, when each of these two glomeruli was selectively activated, flies showed as robust an attraction to vinegar as wild-type flies. Notably, a higher concentration of vinegar excites an additional glomerulus and is less attractive to flies. We show that activation of the extra glomerulus is necessary and sufficient to mediate the behavioural switch. Together, these results indicate that individual glomeruli, rather than the entire pattern of active glomeruli, mediate innate behavioural output.
Movement Intention After Parietal Cortex Stimulation in Humans
Parietal and premotor cortex regions are serious contenders for bringing motor intentions and motor responses into awareness. We used electrical stimulation in seven patients undergoing awake brain surgery. Stimulating the right inferior parietal regions triggered a strong intention and desire to move the contralateral hand, arm, or foot, whereas stimulating the left inferior parietal region provoked the intention to move the lips and to talk. When stimulation intensity was increased in parietal areas, participants believed they had really performed these movements, although no electromyographic activity was detected. Stimulation of the premotor region triggered overt mouth and contralateral limb movements. Yet, patients firmly denied that they had moved. Conscious intention and motor awareness thus arise from increased parietal activity before movement execution.
Recently, Brain Windows mentioned the report A genetically encoded fluorescent reporter of ATP:ADP ratio. We invited Dr. Jim Berg, the lead author of the study to provide additional background to our readers. Below, Jim provides a fascinating look at rationale behind sensor development. I really like that they came at this problem with a biological question in mind, something I would recommend before anyone start the development of a genetically encoded indicator.
A pixel-by-pixel ratio of the 490 nm excitation image by the 430 nm excitation image from two cultured HEK293 cells expressing Perceval during control conditions (left) and after 40 min of metabolic inhibition with 5 mM 2-deoxyglucose (right)
Here’s a little insight into why we decided to develop a fluorescent sensor for cellular energy, and how Perceval evolved. One of the primary research interests of the Yellen lab is the interaction between diet and epilepsy. The ketogenic diet, a high fat, low carbohydrate regimen, is remarkably effective at reducing seizure number. We are investigating how the transition in brain metabolism from glucose to a mixture of glucose and ketone bodies (the metabolically active byproduct of fat metabolism) could lead to a change in neuronal excitability. Previously, we described how acute application of ketone bodies reduces the excitability of substantia nigra neurons, an effect that relies on the opening of ATP-sensitive potassium (KATP) channels. Our hypothesis is that the inhibition of glycolysis by ketone body metabolism leads to a reduction in sub-membrane ATP, resulting in an opening of KATP channels and a decrease in neuronal excitability. This relies on the controversial idea that sub-membrane ATP is provided by glycolysis (possibly by glycolytic enzymes tethered to the membrane), and that the diffusion of ATP is restricted between the submembrane space and bulk cytoplasm, and concept known as “compartmentation”. To fully test this hypothesis, we required an optical sensor for ATP levels.
When planning these experiments, our first thought was to use Luciferase to detect different subcellular ATP levels. For a number of reasons, primarily Luciferase’s weak signal, we decided that a fluorescent sensor for ATP would be much more useful for our application. Our initial approach was a FRET-based design, with CFP and YFP tethered to a bacterial periplasmic binding protein that dimerized upon ATP application. Although these sensors gave some encouraging results, we never got the change in signal that would be required for cellular assays. We then adopted the ‘circularly permuted fluorescent protein (cpFP) approach that had previously produced sensors for calcium (pericam) and hydrogen peroxide (HyPer). We inserted the yellow fluorescent protein cpmVenus into the loop of the bacterial ATP binding protein, GlnK1 (involved in the regulation of ammonia transport) and found that application of small amounts of ATP to the purified sensor led to a substantial change in the excitation spectrum of the sensor. The affinity of the sensor for ATP was extremely high, orders of magnitude more sensitive than would be appropriate for cellular assays. We also found that our sensor responded to ADP application, only with a much smaller fluorescence change. It was then that we determined that these two perceived negatives (too high affinity and ADP binding) would lead to a sensor that reports the ratio of ATP to ADP. In a bit of good fortune, our design for an ATP sensor had in fact given us a sensor for the more valuable ATP:ADP ratio. After tinkering with our sensor by semirandom mutagenesis of the GlnK1 portion of the protein, we expressed the improved sensor, which we named Perceval (for permuted reporter of cellular energy value) into cultured cells and monitored a change in fluorescence with metabolic inhibition.
Right now, we are excited to use Perceval to investigate neuronal/glial metabolism in mammals. We may target subcellular ATP by either tethering Perceval to a membrane protein, or by using TIRF microscopy. In addition, we are continuing to design improved versions of Perceval, as well as sensors for other metabolic intermediates. We also hope that these sensors will be useful in applications beyond neuronal metabolism, from studies of cancer cells to bacterial metabolism.
This coming Tuesday and Wednesday (Feb 17th & 18th) at UCSD, there will be a symposium honoring Roger Tsien, featuring presentations from 32 former and current members of the Tsien Lab. The topics are quite diverse, concentrated in genetically-encoded indicators, but also featuring fluorescent cell penetrating peptides for cancer therapy, photophore ligases for imaging synaptic development, and even a radical new design for the internal combustion engine.
The quality of speakers and subjects looks to be outstanding. Here is a complete schedule. You may notice that at 11:15 AM on Tuesday in Price Center East Ballroom, I will be presenting recent progress we have made in the development of genetically-encoded calcium indicators and their application to in vivo imaging. Don’t miss that one! 🙂 Roger’s talk, which will assuredly be equal parts absorbing, humorous, and illuminating, is at 4pm Wednesday in the Price Center Theater.
If you live in Southern California and are interesting in imaging technology, there isn’t a better place to be than this symposium. If you can’t make it, Brain Windows will have a full write-up following the event.
Here is the un-official schedule.
Tuesday February 17th – Price Center East Ballroom
9:00 -9:05 Varda Levram -Ellisman Opening
9:05-9:15 Palmer Taylor
Designing the next generation of genetically encoded sensors
9:15-9:30 Roger Heim
FRET for compound screening at Aurora/Vertex
9:30-9:45 Amy Palmer
Designing and using genetically encoded sensors: Lessons I learned from Roger
9:45-10:00 Robert Campbell
Beyond brightness: colony screens for fluorescent protein photo stability and biosensor FRET changes
10:00-10:15 Colette Dooley
GFP sensors for reactive oxygen species: Tying up loose ends and looking forward.
10:15-10:30 Peter Wang
Fluorescent Proteins and FRET biosensors for visualizing cell motility and mechanotransduction
Fluorescent proteins in neuroscience
11:00-11:15 Brian Bacskai
Aberrant calcium homeostasis in the Alzheimer mouse brain
11:15-11:30 Andrew Hires
Watching a mouse think: Novel fluorescent genetically-encoded calcium indicators applied to in vivo brain imaging
11:30-11:45 Alice Ting
Imaging synapse development with engineered photophore ligases
11:45-12:00 Rex Kerr
3D calcium imaging in C. elegans
Clinical applications
12:00-12:15 Todd Aguilera
Activatable Cell Penetrating Peptides for use in clinical contrast agent and therapeutic development
12:15-12:30 Quyen Nguyen
Surgery with Molecular Fluorescence Imaging Guidance
Fluorescent probes (Chemistry)
1:30-1:45 Tito Gonzalez
Voltage-Sensitive FRET Probes & Applications
1:45-2:00 Paul Negulescu
From watching ions to moving them
2:00-2:15 Timothy Dore
Roger-Inspired Photochemistry: Releasing Biological Effectors with 2PE
2:00-2:15 Joe Kao
Electron Paramagnetic Resonance Imaging in Living Animals
2:15-2:30 Brent Martin
Chemical probes for studying protein acylation
2:30-2:45 Jianghong Rao
Non-GFP based probes for imaging of the hydrolytic enzyme activity
Cellular research with and without Fluorescent probes
3:15-3:30 Carsten Schultz
Cell membrane repair visualized by GFP fusion proteins
3:30-3:45 David Green
Transcriptomes and Systems Biology: application to early mammalian embryogenesis
3:45-4:00 Clotilde Randriamampita
Paradoxical aspects of T cell activation revealed with fluorescent proteins
4:15-4:30 Wen-Hong Li
Studying dynamic cell-cell communication in vivo by Trojan-LAMP
4:30-4:45 Martin Poenie
Aim and Shoot: Two roles for dynein in T cell effector function
4:45-5:00 Gregor Zlokarnik
From bla to blah, blah in 20 years
5:00-5:15 James Sharp
President, Zeiss MicroImaging Gmbh
February 18 2009 – Leichtag 107
Cellular research with and without fluorescent proteins
9:00-9:15 David Zacharias
Fluorescent Proteins, Palmitoylation and Cancer: two out of three ain’t bad
9:15-9:30 Jin Zhang
Visualization of Cell Signaling Dynamics: A Tale of MAPK
9:30-9:45 Paul Sammak
Nuclear organization and movement in pluripotent stem cells measured by Histone GFP H2B
Branching out
9:45-10:00 Yong Yao
NIH Toolbox Program
10:00-10:15 Oded Tour
The Tour Engine – A novel Internal Combustion Engine with the potential to boost efficiency and cut emissions
Into the future
10:45-11:00 Xiaokun Shu
Visibly and infrared fluorescent proteins: photophysics and engineering
11:00-11:15 Michael Lin
Engineering fluorescent proteins for visualizing newly synthesized proteins and improving FRET-based biosensors
11:15-11:30 Jeremy Babendure
Training our next generation of Fluorescent Protein Enthusiasts
Main Event – Price Center Theater
4:00-5:00 Roger Tsien
Chancellor invitational lecture 2008 Nobel Prize in Chemistry
Today, Brain Windows welcomes its first guest contributor. Dr. Jasper Akerboom is a post-doctoral associate in the lab of Loren Looger at Janelia Farm, and is the lead author on a recently published report on the structure of the genetically-encoded calcium sensor, G-CaMP2. We are very grateful for his contribution!
After the previous post describing G-CaMP2 crystallization two papers describing the crystallization and structure determination appeared online:
Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. Akerboom J, Vélez Rivera JD, Rodríguez Guilbe MM, Alfaro Malavé EC, Hernandez HH, Tian L, Hires SA, Marvin JS, Looger LL, Schreiter ER. J Biol Chem. 2008 Dec 18.
and
Structural Basis for Calcium Sensing by GCaMP2. Wang Q, Shui B, Kotlikoff MI, Sondermann H.Structure. 2008 Dec 12;16(12):1817-27.
Both papers are very similar, with minor differences in the approach of some of the problems, which will be described below.
In the paper of Wang et al., crystallization of GCaMP2 is achieved by removing the pRSET tag, important for in vivo GCaMP2 function (Nakai et al). Removal of disordered expression tags often is essential for protein crystallization, however, Akerboom et al crystallized GCaMP2 with this tag still present. Spectrophotometric properties of purified GCaMP2 protein with and without pRSET module are identical.
Both in the JBC paper as well as the Structure paper the authors describe the presence of dimeric calcium loaded GCaMP2, appearing as a minor fraction during gel filtration analysis.
Size-exclusion trace of calcium loaded (blue line) and calcium free (red line) G-CaMP2
Akerboom et al initially only crystallized the dimeric form of GCaMP2. Attempts to crystallize monomeric GCaMP2 failed. Selection and mutagenesis of amino acids partaking in the dimer interface in GCaMP2 resulted in the subsequent crystallization of monomeric GCaMP2. Wang and coworkers were able to crystallize both forms without mutagenesis, although their GCAMP2 molecule had its pRSET module removed, indicating a potential role for the pRSET peptide in dimerization.
Both monomeric and the dimeric crystal forms described in both papers are essentially the same.
Dimeric G-CaMP2
Monomeric G-CaMP2
The dimeric form of G-CaMP2 is a domain swapped dimer with the M13 peptide (magenta) of each monomer bound by the calcium loaded CaM domain (cyan) of the other. The monomer is very different from the dimer, with the M13 peptide bound by the CaM domain of the own molecule. The interface between CaM and cpEGFP is considerably different between the two different oligomeric states of G-CaMP2.
Wang hypothesizes about a potential role of residue T116 (T203 in GFP numbering) playing in chromophore stabilization in calcium saturated G-CaMP2; this residue adopt a different rotamer in the dimeric structure, in a way that this threonine cannot partake in the hydrogen bond network, dimeric G-CaMP2 is less bright. In the paper by akerboom et al this residue adopts double conformations, so its not clear if this residue is actually the reason for this effect. In addition the mutation T203V results in increased fluorescence in G-CaMP2. Valine is hydrophobic and cannot participate in hydrogen bond formation at all.
Both groups performed mutational analysis of G-CaMP2. Both groups actually described a few identical positions (R81 and R377), and came roughly to the same conclusions, R81 and R377 play a role in the calcium loaded state of the protein. Wang et al performed the experiment using both mutations, and showed a profound decrease of fluorescence.
The group from Janelia Farm made some efforts to improve sensor functionality, and showed that replacing an aspartate close to the chromophore in the calcium saturated state with a tyrosine increases fluorescence by lowering the percentage of protonated chromophore.
Both Wang et al and Akerboom et al tried to study apo-G-CaMP2. Wang and co-workers used small-angle X-ray scattering (SAXS) of apo-G-CaMP2 and solved the structure of cpEGFP. The other group mutagenised all four EF-hands of CaM, removing the calcium binding capacity of G-CaMP2, and subsequently crystallizated the calcium binding deficient G-CaMP2. Both SAXS and crystallization indicated a more open structure of GCaMP2 compared to the calcium loaded state.

SAXS with fitted cpEGFP and 3CLN structures
apo G-CaMP2 structure
In the crystal structure, the M13 peptide and the C-terminal domain of CaM are disordered, indicating the large degree of freedom in apo G-CaMP2. Part of the linker between the M13 peptide and cpEGFP in the apo structure forms part of the beta barrel of cpEGFP.
Both papers will contribute to the understanding of the GECI G-CaMP2. Further directed mutagenesis studies on the basis of the results described in both manuscripts will hopefully result in a better sensor for in vivo imaging.
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