Cameleon-Nanos : High Affinity GECIs

Takeharu Nagai’s lab has published in Nature Methods, Spontaneous network activity visualized by ultrasensitive Ca²⁺ indicators, yellow Cameleon-Nano, demonstrating a new set of calcium indicators based on yellow cameleon. Back when he was still Take-san, Take’s ability to churn out and manually screen hundreds of cameleon variants was impressive and inspiring. With high-throughput GECI pipelines now ramping up at Janelia, the idea of laboriously screening 200 variations on a theme (be it cameleons or GluSnFRs), seems a bit archaic. However, this paper is a good example of the progress that can still be made by understanding the needed sensor parameters and fiddling with the primary amino acid structure in a relatively low-throughput way. Take-sensei’s results are another example of the pramatic rule in protein design, “when in doubt, tinker with the linker.”

The cameleon-nano family achieves greater apparent calcium affinity than YC2.60-4.60, reaching levels of up to 15nM.  They did this by increasing the flexibility of the linker by extending the standard Tsien/Miyawaki/Baird Gly-Gly-Ser linker with additional glycines.  In this case, the longer the linker between the CaM and M13 segments, the greater the apparent affinity. Interestingly, improvement by increasing linker flexibility is precisely the opposite the advice Atsushi and Take gave me for achieving high ratio changes with FRET reporters.  Back at RIKEN in 2002, they suggested I use short, stiff linkers to restrict the rotational freedom of the fluorescent pairs.  Then one could find orientations where relative rotation of dipole moment gave much greater FRET changes than would be expected from changes in FP distance alone. Take and Atsushi’s big YC2.60/3.60 paper strongly supported this idea!  However, as our understanding of the ideal parameters of calcium sensor’s for in vivo imaging has grown, development directions have adjusted.

Cameleon-Nanos achieve higher signal/noise for sparse action potentials at the expense of linearity.  Like Fluo-4, the signal saturates at relatively low AP frequencies.  I think the absolute affinities measured for this family (15, 30, 50 and 140nM) should be considered very rough estimates. They extrapolated these values from stopped-flow binding experiments, because

Although we would like to measure the koff of YC2.60 and its high affinity variants such as YC-Nano15, we could not do it because it was very difficult to precisely control free Ca2+ concentration at around few tens of nM as far as we used EGTA (Kd for Ca2+ = 151 nM in 0.1 M ionic strength, pH 7.2 at 25 oC). For this purpose, much stronger Ca2+ chelator with a smaller Kd value was required. However there is no such Ca2+ chelator available now.

I’m not sure why they didn’t just use the higher affinity, Mg++ insensitive, chelator BAPTA to make the Kd measurements the right way, with a linear regression of log-log fluorescence/concentration values.  Due to instrument dead time, and the high affinity, I didn’t like stopped-flow based Kd measurements in the early GCaMP papers, and I don’t like them now.  Also, the apparent calcium Kd will be highly dependent on solution ionic strength and [Mg++] which is unreported. Despite these quibbles, which are important only inasmuch as they give insight into the mechanism of improvement and the direction of future development, the cameleon-nano family looks promising for mammalian brain imaging.  I still wonder if, assuming the reported Kd values are relevant in vivo, YC2.60 would be the best of the bunch, since cortical neurons have a resting Kd of ~50nM, which implies that a single AP transient of say 200nM free [Ca++] increase would push the calcium levels right up into the sweet-spot of YC2.60’s sensitivity.

This is all the more interesting given the recent results in YC3.60 imaging from Maz Hasan’s group.  Previously, he had shown that transgenic YC animals were pretty bad for imaging.  However, AAV-mediated gene delivery of YC3.60 has significantly improved the responses of the YC family. I’m not sure if they are really up to GCaMP3 levels under identical in vivo conditions, but they might have better long-term protein stability (or that might depend on which viral serotype is used.) What about cameleon-nanos, what about YC2.60?

Infrared fluorescent proteins

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, mPlum, mKate, 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.

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

 

IFP1.4 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.

Symposium : A Revolution in Fluorescence Imaging

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

Preview : Structure of G-CaMP2

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, = 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…

Structural Basis for Calcium Sensing by GCaMP2

Qi Wang¹,Bo Shui²,Michael I. Kotlikoff²andHolger Sondermann^1,,

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.

Some interesting posters @ SfN

Here’s a few posters that caught my eye at SfN.  Click the meeting planner for the full abstract

Optimizing two-photon activation of channelrhodopsin-2 for stimulation at cellular resolution

J. P. RICKGAUER^1,2, D. W. TANK^1,2; 

Spiral pattern of 2-photon excitation can drive neurons to spike.  A low NA objective helps. Need to do piezo-based Z-scanning if you use high NA, don’t with low NA.

In vivo two-photon imaging 1 mm deep into cortical brain tissue with novel microprism probe 

*T. H. CHIA, M. J. LEVENE; 

A cute method to image 1mm into cortex with 2-photon imaging. They used 2-6 month old mice. The just took a triangular prism whose hypotenuse was silvered and stuck it in the cortex. Then they internally reflected the beam off the prism and fired it sideways into cortex. Got good SNR to 300um lateral distance.  Some clippling of beam at edges of the prism gave somewhat inconsistent spatial resolution.

Self-complementary adeno-associated viral vectors for fast, efficient labeling of neurons and astrocytes in visual cortex in vivo

R. L. LOWERY¹, Y. ZHANG², C. LAMANTIA¹, B. K. HARVEY³, A. K. MAJEWSKA¹; 

AAV is the way to go for expression of GECIs and ChR2 in vivo, but it takes a long time to express at high levels (2 weeks). They show that using a double stranded DNA version of AAV rather than single stranded gets protein expression up high much faster. Very high expression after one week. This is because the virus doesn’t need to take the time to make the second strand before expressing the protein.  See Xiao, X J. Virol 1998

Detection of single action potentials in vitro and in vivo with genetically-encoded Ca²⁺ sensors

S. MEYER ZUM ALTEN BORGLOH¹, D. J. WALLACE², S. ASTORI³, Y. YANG³, M. BAUSEN³, S. KUGLER⁴, M. MANK⁵, O. GRIESBECK⁵, J. NAKAI⁶, A. MIYAWAKI⁶, A. E. PALMER⁷, R. Y. TSIEN⁷, R. SPRENGEL³, J. N. D. KERR², W. DENK³, M. T. HASAN³; 

Everything in the poster was in the Nature Methods paper.  Conversation reveled that YC3.60 works as well or better than D3cpv. Only have done up to whisker evoked stimulation, no imaging of spontaneous YC3.60 signals yet.

Characterization of improved probes for the hybrid voltage sensor method of voltage imaging

D. WANG¹, Z. ZHANG², B. CHANDA¹, M. B. JACKSON¹; 

A nice little sensor optimization poster.  They took the hVOS hybrid voltage sensor of dipicrylamine with membrane tethered GFP and improved it by changing the chromophore to Cerulean, and by using the “membrane-staple” strategy. Having membrane anchors on both the N and C-termini gave better quenching. Fast response, ~0.5ms, and 20% dF/F.

Crystal structure of the genetically encoded calcium indicator gcamp2

*J. AKERBOOM¹, L. TIAN¹, S. VISWANATHAN¹, S. A. HIRES¹, J. S. MARVIN¹, E. R. SCHREITER², L. L. LOOGER¹; 

Jasper made crystal structures of G-CaMP2 in the apo and bound states.  Bound states crystalized as a heterodimer, but he was able to also crystalize the monomer. The structures show a pore to the chromophore in the apo state that is plugged in the Ca-bound state. Thus, the quenched apo state is due to solvent access to the chromophore.  This structural data should help rational design of better G-CaMP sensors.

A brief history of calcium imaging

A few months ago I threw together a short presentation on the history of calcium imaging for a journal club here at Janelia. It is incomplete. It lacks notes. It is technical. It focuses much attention on early genetically-encoded indicators. However, calcium imaging is so intertwined with the work of Roger Tsien, my Ph.D. thesis advisor, and since he just won the Nobel Prize, I thought it might be of interest to some of the audience of Brain Windows. It does provide a little bit of background for some of the more recent developments chronicled on this site.

Enjoy.

2008 Nobel Prize in Chemistry to GFP

This morning, the Nobel committee recognized the work of Osamu Shimomura, Martin Chalfie and Roger Tsien “for the discovery and development of the green fluorescent protein, GFP” by awarding them the Nobel Prize in Chemistry for 2008.  A video of a great lecture on fluorescent proteins by Roger Tsien is available here.

Green Fluorescent Protein

Shimomura first discovered GFP during the study of the bioluminescent protein aequorin, the mechanism by which certain jellyfish glow.  In the footnote to his seminal paper on aequorin purification, he noted the additional presence of “a protein giving solutions that look slightly greenish in sunlight through only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite, has also been isolated from squeezates.” 

Aequorea Victoria

Chalfie took the cDNA of GFP and first expressed it bacteria and worms.  He demonstrated GFP could be used as a molecular tag. Surprisingly, the protein folded and functioned without the use of co-factors specific to the jellyfish.

Tsien developed GFP into the many useful variants we use today.  He reported the S65T point mutation that greatly improved its fluorescent characteristics. His lab also evolved GFP into many other color variants, and demonstrated that these variants could be used as genetically-encoded intracellular sensors for calcium, enzyme action, and glutamate.

Chromophore of the S65T mutant of GFP

The odd man out in this triumvirate is Douglas Prasher.  With a tiny lab and budget, Prasher discovered the primary sequence of GFP and cloned the cDNA of GFP. Unfortunately, around the time of his work’s publication, his grant ran out. Prasher sent out DNA samples to Chalfie, Tsien and others, shut down his lab and left science. Prasher’s contribution was the essential foundation for the explosion of developments in the field.

Some argue that Tsien would have already won the Nobel prize for calcium signaling if not for his contribution to GFP. As a graduate student, he invented the high affinity calcium chelator BAPTA. Using BAPTA as a foundation, he created a large family of fast, bright calcium dyes, including fura-2.  Nearly every fluorescent dye for calcium was either his invention or a close variant of one of these. The importance of these tools for understanding intracellular communication cannot be overstated.

Transgenic GFP mouse