Endogenous Dark Chromophore Imaging via Modulated Stimulated Emission

11 11 2009
Here is an interesting paper, Imaging chromophores with undetectable fluorescence by stimulated emission microscopy, from Sunny Xie’s group.  They pump the sample with a excitation laser while simultaneously hitting it with a longer wavelength laser to induce stimuated emission. The pump laser is modulated at a high frequency which they can pick up and amplify with a lock-in amplifier.
2009-03-03106D-2009-03-03106_figure_1.ppt

Theory and illumination schematic

2009-03-03106D-2009-03-03106_figures_4.ppt

In two examples of imaging from a mouse ear, (above) shows the distribution of TBO, a photodynamic therapy drug, following drug administration, (below, red) shows the distribution of hemoglobin in blood vessles.

How specific is the detection of endogenous chromophores?  They report that 60nm is the absolute detection limit, but this is for a pure chromophore in water.  In real cells there will be many other endogenous chromophores at various concentrations. For example, endogenous background fluorescence of flavins is often easily seen when imaging at CFP wavelenghts. Watt Webb has been imaging those types of chromophores for years. The intersection of both a preferred excitation wavelengh and a preferred stimulated emission wavelength will provide some selectivity, but I suspect this will be most useful for imaging the distribution of fairly highly expressed chromophores in vivo. Distinguishing chromophores with highly overlapping spectra may not be possible.  Of course, many, many proteins don’t have distinctive chromophores (tyrosine does not count!) built in to them, so GFP won’t be out of work any time soon.  However, this stimulated emission imaging doesn’t require transgenic or small molecule labeling, so it could potentially allow imaging in humans.

2009-03-03106D-2009-03-03106_figure_2.ppt

Do the absorbance and emission spectra and the excited state lifetime provide sufficient selectivity to detect low concentrations of chromophores in vivo?

Thanks goes to Reporter Gene for the story tip.`





Automated ROI analysis for calcium imaging

2 10 2009

One of the most time consuming and frustrating tasks associated with fluorescence imaging in the brain is picking out your regions of interest.  Which pixels do you include in as part of the cell and which are part of the surrounding neuropil?  Often, the answer is not obvious, and even with painstaking selections you can make errors.  Eran Mukamel et. al, from Mark Schnitzer‘s lab just published this Neurotechnique Automated Analysis of Cellular Signals from Large-Scale Calcium Imaging Data that aims to simplify and improve the results of ROI selection. 

The authors used a multistage approach to identify and quantify the calcium-dependent fluorescence changes of imaged neurons. First, they used principal component analysis to identify the components of the image that were likely calcium signal related and which were noise.  The sparse nature of the calcium response (calcium transients are brief and spatially confined) helped the separation from the noise. They threw the noise away.  Then they used independent component analysis to pick out which components of the calcium signal changed in a manner independent from other pieces of the signal.  These likely represent individual cells. Using this output, they performed auto-segmentation of the image into numerous individual neurons or processes and measured the fluorescence change in those regions.  In simulations of data, it resulted in superior data fidelity over hand drawing ROIs.  They also validated it with real in vivo calcium imaging.

 

Automated Cell Sorting Identifies Neuronal and Glial Ca2+ Dynamics from Large-Scale Two-Photon Imaging Data

Automated Cell Sorting Identifies Neuronal and Glial Ca2+ Dynamics from Large-Scale Two-Photon Imaging Data

 

Whether its neuronal imaging, high-speed motion tracking or multielectrode recordings, tremendously large data sets are currently being generated in systems neuroscience. It is simply impossible for a single post-doc to crunch all of her data without major automated computational techniques.  In calcium imaging, the resources that have been poured into the development and release of powerful new tools requires an equal effort on the data analysis end to maximize the value of this technique.  The automated algorithms presented in this paper look very promising and we will definitely be checking them out in the near future.





Photoactivated Transcription Revisted

14 07 2009

Looks like there has been some new results in the field of photoactivated transcription.  Unlike the fully genetically-encoded systems reviewed in a Journal Club, this uses a hybrid genetic and small molecule approach. In Doxycycline-dependent photoactivated gene expression in eukaryotic systems, Cambridge et al. add the photolabile protecting groups to doxycyclin derivatives, which then function as photoactivatable switches in the commonly used Tet-on system. Dr. Dan O’Connor described the technique as “the path of least resistance to photoactivated transcription.” 

 

Local (left) and global (right) GFP expression following optical uncaging of cyanodoxycyclin

Local (left) and global (right) GFP expression following optical uncaging of cyanodoxycyclin

 

 

      The authors were able to get robust gene expression with standard UV irradiation, but also were able to uncage sufficient cyanodoxycycline with two-photon illumination to cause highly localized gene expression in cultures.  In live tadpoles, they stuck to UV for the greater efficiency.

     The standard caveats of the tet system apply. The off-state still has a bit of residual gene expression, which is fine for some applications (like fluorescent tagging), and a dealbreaker for others (cre induction). Drug delivery takes time and comes with diffusion, penetration and clearance issues.  UV penetration through deep tissue is going to be a big technical hurdle to overcome to apply this to full-grown mammals. Blasting living tissue with high power UV usually isn’t a good idea. Despite these caveats, the system clearly works and I’d bet the authors are already applying the system to some next-step applications and biological questions. The potential of selectively turning on genes in functionally identified neurons via light is enormous.  It is one of the most likely eventual avenues into possible optical activation or suppression of specific thought patterns (at least if you are willing to squirt virus into your brain and eat a bunch of nasty antibiotics).





Journal Scan – Transynaptic tracing, fly olfaction, fast super-resolution, localization of perception

8 05 2009

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.





Infrared fluorescent proteins

8 05 2009

Hunting for new fluorescent proteins in the coral reefs of the Caribbean and Australia is a task that a lucky few researchers have managed to get funding for. Scuba diving in some of the world’s most beautiful places; it’s not a bad gig, if you can get it.  Most fluorescent protein scientists are confined to a lab, mutating existing fluorescent proteins from jellyfish and coral. Shifting their excitation and emission spectra has allowed multiple fluorescent proteins to be used as molecular highlighters at the same time, since their colors are distinct from each other. Some members of this palette are shown in Brain Windows top image bar.  After over a decade of research, the spectrum is pretty well covered.  Except for one area…  The infrared.

The near-infrared band is an area of enormous importance to scientific researchers, because is contains the spectral window where the body becomes transparent. Hemoglobin in the blood strongly absorbs visible wavelengths shorter than 650nm, while water absorbs wavelengths above 900nm.  If a fluorescent protein could be found, or engineered to have excitation and emission within this window, we could use it to peer much deeper into the body. The near-IR light penetrates much more easily into and out of the tissue.  This is easily seen by pressing a flashlight against your hand.  Only the deep red light passes through. The quest for an infrared fluorescent protein has preoccupied several labs for a decade.

Efforts to push FPs out to the infrared resulted in mCherry, mPlummKate, among others.  The further red-shifting of these proteins is constrained by the space limitations of the beta-barrel structure of GFP-like proteins.  In general, the longer the resonance chain of the chromophore, the longer the wavelength of the chromophore’s excitation and emission spectra.  It has been difficult to extend the FP spectra beyond 650nm without adding an additional bond to the resonance structure, for which there is little space left in the protected center of the barrel.  

 

Wavelength of excitation and emission is longer in larger resonance structures.

Wavelength of excitation and emission is longer in larger resonance structures.

In Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome Shu et al. of Roger Tsien’s lab, looked beyond traditionally used fluorescent proteins to extend the palette into the near-IR.  They engineered a protein, IFP, which binds biliverdin, a natural product involved in aerobic respiration (and similar in structure to the phytocyanobilins discussed in our Photoactivated Transcription journal club post). Biliverdin is non-fluorescent in solution, but when bound to IFP, it is rigidized and becomes fluorescent, with excitation at 684nm and emission at 708nm.  IFP can then be fused to the protein of interest and visualized through thick absorbent tissue.  Even the liver, which is dense with heme, is easily seen through the skin when labelled with IFP.  

 

IFP1.1 expressed in mouse liver is clearly visible through the skin 

 

IFP1.1 expressed in mouse liver is clearly visible through the skin

 

IFP1.4 should immediately push forward the field of in-vivo imaging of cancer and other diagnostics, at least in animal models.  It’s not clear yet how useful it will be for in-vivo brain imaging, they show cultured neurons expressing IFP1.4 become fluorescent upon biliverdin application, but can biliverdin be effectively delivered to neurons in vivo? Like channelrhodopsin, there may be sufficent amounts of endogenous co-factor to make the protein useful without exogenous application. Perhaps of greater importance is the new engineering avenues IFP opens up.  This is an entirely new, two-component scaffold, with different characteristics from GFP that protein engineers will be able to optimize and exploit.   Over the next decade, IFP may spawn as diverse a set of tools as GFP has over the previous one.





Background : Perceval, the ATP:ADP sensor

12 03 2009

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)

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.





Symposium : A Revolution in Fluorescence Imaging

11 02 2009

header-jellyfish

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






Update : Structure of G-CaMP2

12 01 2009

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.
image001
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

Dimeric G-CaMP2

Monomeric 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

SAXS with fitted cpEGFP and 3CLN structures

 apo G-CaMP2 structure

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.





BrainStorm 1 : The Calcium Memory Sensor

9 01 2009

As mentioned in the previous post, this is the first installment of BrainStorm, a section of ideas I have under development, but don’t have the time to physically work on.  This section will contain organically developed ideas, organized by project.  Reader feedback is encouraged.

How can we identify the group of neurons that encode a particular thought?  

I don’t want to simply see correlations of in activity of a few scattered neurons with a given thought, but identify the entire neuronal ensemble.  Which neurons are active at a precise moment in a task?  How are they wired together? Which are the drivers of activity?

Existing technology is inadequate to identify the entire neural ensemble that encodes a thought. Immediate early gene expression  patterns have not been shown to be precisely correlated with brain activity, and have a temporal resolution on the order of minutes. Genetically encoded calcium sensors (GECIs) have the necessary temporal and spatial resolution, but their response is nearly as fleeting as a thought, making it impossible to simultaneously record from networks of thousands of possible participants with current microscopy techniques.

In BrainStorm 1, I will outline a technology, photoswitchable genetically-encoded calcium memory sensors, that can identify all the neurons in a large network that are active during user-specified, aribitrarly brief or long time periods.  I will propose four potential strategies for construction of these sensors, and detail practical considerations for sensor design, screening and application.





Preview : Structure of G-CaMP2

10 12 2008

A high-resolution crystal structure of the genetically-encoded calcium indicator G-CaMP2 would aid in rational design of improved calcium indicators. Crystallization of G-CaMP2 was first reported here :

Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

M. M. Rodríguez Guilbe, E. C. Alfaro Malavé, J. Akerboom, J. S. Marvin, L. L. Looger and E. R. Schreiter

Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, [beta] = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.

High-resolution atomic structures and mutational analysis were presented at SfN 2008 (see this previous post)

However, today a competing group has published an independent report on a similar set of G-CaMP2 structures in Cell Structure.  More details to come…

picture-7

Structural Basis for Calcium Sensing by GCaMP2

Qi Wang1,Bo Shui2,Michael I. Kotlikoff2andHolger Sondermann1,Go To Corresponding Author,

Genetically encoded Ca2+ indicators are important tools that enable the measurement of Ca2+ dynamics in a physiologically relevant context. GCaMP2, one ofthe most robust indicators, is a circularly permutated EGFP (cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for studying Ca2+ fluxes invivo in the heart and vasculature of transgenic mice. Here we describe crystal structures of bright and dim states of GCaMP2 that reveala sophisticated molecular mechanism for Ca2+ sensing. In the bright state, CaM stabilizes the fluorophore in an ionized state similar to that observed in EGFP. Mutational analysis confirmed critical interactions between the fluorophore and elements of the fused peptides. Solution scattering studies indicate thatthe Ca2+-free form of GCaMP2 is a compact, predocked state, suggesting a molecular basis for the relatively rapid signaling kinetics reported for this indicator. These studies provide a structural basis for the rational design of improved Ca2+-sensitive probes.