Spying on cGMP with FRET
2
NATURE METHODS | VOL.3 NO.1 | JANUARY 2006 | 11 NEWS AND VIEWS Spying on cGMP with FRET Kees Jalink A new carefully optimized and characterized genetically encoded fluorescent sensor for cyclic GMP (cGMP) has fast kinetics and properties that should make it an excellent compromise between sensitivity and specificity when compared to existing sensors. Cells have a vast array of surface receptors that act as antennas to pick up messages from the outer world. In the cell interior, activated receptors relay their message through a lim- ited set of secondary signals (‘second mes- sengers’), raising the question of how the specificity of the message is retained. It is commonly hypothesized that precise timing and localization of the intracellular signals is part of the answer, but traditional biochem- ical assays are not suited to studying these phenomena. Consequently, researchers have devised so-called fluorescent sensors, that is, dyes whose fluorescent properties are altered upon binding their cognate target molecule. When used in conjunction with fluorescent microscopy this allows the dynamic readout of second messengers with high spatiotem- poral precision. Recently, genetically encoded sensors were developed. These typically consist of a protein that changes its conformation upon binding to the messenger and an optical ‘trick’ to visualize this shape change, most commonly fluorescence resonance energy transfer (FRET) between attached donor and acceptor fluorescent proteins (Fig. 1). In the last several years, FRET sensors for Ca 2+ , IP 3 , cyclic nucleotides, protein phosphory- lation and more have been developed, and researchers in many laboratories continue to screen for new and improved sensors. In this issue of Nature Methods, Lohse and colleagues report the results of an extensive study aimed at optimizing FRET sensors for cGMP 1 . cGMP is an important second messenger controlling diverse functions such as smooth muscle relaxation, photo- transduction, gene expression, ion channel gating and synaptic plasticity. The authors tested a variety of cGMP-binding domains from different proteins by sandwiching them between enhanced cyan (CFP) and yellow (YFP) fluorescent proteins. Several of those chimeras showed FRET changes when the cGMP concentration was ele- vated in HEK293 cells. The authors then proceeded to characterize the constructs for affinity, selectivity against cyclic AMP (cAMP), fluorescence ratio change and kinetic parameters. Notably, also included in this characterization were the two cGMP sensors previously reported by the groups of Umezawa and Dostmann 2,3 . One of the new sensors, cGES-DE5, a construct consisting of the regulatory GAF domain from phos- phodiesterase 5 flanked by enhanced CFP and YFP stood out as particularly favorable. Compared to other sensors, cGES-DE5 had an impressive cGMP-induced FRET change and much faster response times. With other properties tested being comparable to those of the best existing sensors, cGES-DE5 is likely to become the construct of choice for future studies. The development of such constructs is labor-intensive, with each success matched by many failures. But the importance of having good sensors for the study of sec- ond messengers cannot be overestimated. It was the development of excellent fluo- rescent Ca 2+ dyes that opened our eyes to the wonderfully complex behavior of the intracellular messenger Ca 2+ . The spatio- temporal concentration of Ca 2+ exhibits a range of manifestations including spikes, Kees Jalink is at the Netherlands Cancer Institute, Division of Cell Biology, Amsterdam, Netherlands. e-mail: [email protected] YFP CFP cGMP Bound to cGMP YFP CFP Unbound Figure 1 | A FRET-based sensor for reporting cGMP dynamics in living cells. Cytosolic cGMP concentration can be assayed as a change in emitted light color when cells expressing cGES-DE5 are excited by 430-nm (violet) light. The sensor consists of the cGMP-binding GAF domain from phosphodiesterase 5 (orange), sandwiched between donor (CFP, cyan) and acceptor (YFP, yellow) fluorescent proteins. Binding to cGMP changes the conformation of the GAF domain, resulting in increased FRET between CFP and YFP. The sizes of yellow and cyan arrows represent the relative amounts of donor and acceptor emission. Katie Ris © 2006 Nature Publishing Group http://www.nature.com/naturemethods
Transcript of Spying on cGMP with FRET
NATURE METHODS | VOL.3 NO.1 | JANUARY 2006 | 11
NEWS AND VIEWS
Spying on cGMP with FRETKees Jalink
A new carefully optimized and characterized genetically encoded fluorescent sensor for cyclic GMP (cGMP) has fast kinetics and properties that should make it an excellent compromise between sensitivity and specificity when compared to existing sensors.
Cells have a vast array of surface receptors that act as antennas to pick up messages from the outer world. In the cell interior, activated receptors relay their message through a lim-ited set of secondary signals (‘second mes-sengers’), raising the question of how the specificity of the message is retained. It is commonly hypothesized that precise timing and localization of the intracellular signals is part of the answer, but traditional biochem-ical assays are not suited to studying these phenomena. Consequently, researchers have
devised so-called fluorescent sensors, that is, dyes whose fluorescent properties are altered upon binding their cognate target molecule. When used in conjunction with fluorescent microscopy this allows the dynamic readout of second messengers with high spatiotem-poral precision.
Recently, genetically encoded sensors were developed. These typically consist of a protein that changes its conformation upon binding to the messenger and an optical ‘trick’ to visualize this shape change, most
commonly fluorescence resonance energy transfer (FRET) between attached donor and acceptor fluorescent proteins (Fig. 1). In the last several years, FRET sensors for Ca2+, IP3, cyclic nucleotides, protein phosphory-lation and more have been developed, and researchers in many laboratories continue to screen for new and improved sensors.
In this issue of Nature Methods, Lohse and colleagues report the results of an extensive study aimed at optimizing FRET sensors for cGMP1. cGMP is an important second messenger controlling diverse functions such as smooth muscle relaxation, photo-transduction, gene expression, ion channel gating and synaptic plasticity. The authors tested a variety of cGMP-binding domains from different proteins by sandwiching them between enhanced cyan (CFP) and yellow (YFP) fluorescent proteins. Several of those chimeras showed FRET changes when the cGMP concentration was ele-vated in HEK293 cells. The authors then proceeded to characterize the constructs for affinity, selectivity against cyclic AMP (cAMP), fluorescence ratio change and kinetic parameters. Notably, also included in this characterization were the two cGMP sensors previously reported by the groups of Umezawa and Dostmann2,3. One of the new sensors, cGES-DE5, a construct consisting of the regulatory GAF domain from phos-phodiesterase 5 flanked by enhanced CFP and YFP stood out as particularly favorable. Compared to other sensors, cGES-DE5 had an impressive cGMP-induced FRET change and much faster response times. With other properties tested being comparable to those of the best existing sensors, cGES-DE5 is likely to become the construct of choice for future studies.
The development of such constructs is labor-intensive, with each success matched by many failures. But the importance of having good sensors for the study of sec-ond messengers cannot be overestimated. It was the development of excellent fluo-rescent Ca2+ dyes that opened our eyes to the wonderfully complex behavior of the intracellular messenger Ca2+. The spatio-temporal concentration of Ca2+ exhibits a range of manifestations including spikes,
Kees Jalink is at the Netherlands Cancer Institute, Division of Cell Biology, Amsterdam, Netherlands.e-mail: [email protected]
YFP
CFP
cGMP
Bound to cGMP
YFP
CFP
Unbound
Figure 1 | A FRET-based sensor for reporting cGMP dynamics in living cells. Cytosolic cGMP concentration can be assayed as a change in emitted light color when cells expressing cGES-DE5 are excited by 430-nm (violet) light. The sensor consists of the cGMP-binding GAF domain from phosphodiesterase 5 (orange), sandwiched between donor (CFP, cyan) and acceptor (YFP, yellow) fluorescent proteins. Binding to cGMP changes the conformation of the GAF domain, resulting in increased FRET between CFP and YFP. The sizes of yellow and cyan arrows represent the relative amounts of donor and acceptor emission.
Kat
ie R
is
©20
06 N
atur
e P
ublis
hing
Gro
up
http
://w
ww
.nat
ure.
com
/nat
urem
eth
od
s
12 | VOL.3 NO.1 | JANUARY 2006 | NATURE METHODS
NEWS AND VIEWS
microRNA detection comes of ageScott M Hammond
The short nature of microRNAs (miRNAs) has presented unique obstacles to experimental biologists. Two research papers in this issue of Nature Methods describe solutions to some of these problems and provide high-resolution data on the expression patterns of these tiny regulatory RNAs.
The explosion of interest in miRNAs has created demand for experimental tools to study these small RNAs. Researchers from all areas of cell and developmental biology want to know the contribution of miRNAs to the regulation of the genes they study. Two research papers in this issue of Nature Methods describe strategies for high-resolution, high-specificity analysis of miRNA expression1,2.
miRNA genes are similar to conven-tional, protein-coding genes; they are tran-scribed by RNA polymerase II, spliced and
polyadenylated3. Here the similarity ends, however. miRNA genes do not code for pro-tein; rather, they contain a stem-loop struc-ture that serves as an endogenous trigger of the RNAi pathway. The stem-loop is excised by the endonuclease Drosha to generate the ‘precursor’. This liberated stem-loop is exported to the cytoplasm where it is fur-ther processed by the endonuclease Dicer to generate a ‘mature’ miRNA of approxi-mately 22 nucleotides (nt). The mature miRNA sequence determines which mRNAs are controlled by the miRNA. Hundreds of
Scott M. Hammond is in the Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.e-mail: [email protected]
miRNAnorthern blot
1993First miRNA
characterized(lin-4)
2001Hundreds of
novel miRNAsuncovered
2002First targetpredictionalgorithms
22 nt
70 nt
miRNAmicroarray
Femtomoledetection
In situhybridization
2004First biologicalrole in humanscharacterized
2005First casual
link to cancer
Figure 1 | Timeline for microRNA detection methods. Initial expression analysis was performed by northern blotting. Experimental approaches progressed through several generations of microarray strategies and have reached an apex with single molecule counting and in situ hybridization.
oscillations, sparks and spirals4 that are believed to have important consequences for cell physiology5,6. These dyes also trig-gered development of additional tools such as ultraviolet light–releasable ‘caged’ Ca2+ and of advanced imaging equipment, and they fuelled thousands of studies that in due course made Ca2+ the ‘queen of messengers’. Notably, recent studies using FRET sensors show that oscillatory changes also take place in the cytosolic level of the related messen-ger cAMP7 and, for example, in phosphory-lation by protein kinase C (ref. 8) suggesting that Ca2+ may not be so outstanding.
Nevertheless, the discoveries made thus far with the new genetically encoded sen-sors clearly fall short of those obtained using fluorescent dyes in the Ca2+ field. It should be realized, however, that the current ‘first-generation’ FRET sensors do not exhibit the favorable properties we have grown used to from modern Ca2+ dyes, including very large signals, excellent signal-to-noise ratios, millisecond response times and the wide selection of affinities and colors neces-sary to match the dye with the experimental requirements.
Intense use has revealed any potential pit-falls of the Ca2+ dyes, and several studies are devoted solely to the development of work-arounds or to the detailed characterization of these dyes. Consequently, the dose-response relationship, kinetics, fluorescent properties, selectivity and biological side effects such as cytotoxicity and buffering of Ca2+ dyes are well-documented. In contrast, most of the first-generation genetically encoded sensors have modest signals, which impedes, for example, their use in screening applications. Furthermore, they have mostly been used in just a handful of studies, and it is seldom that scientists find the time to rigorously test their tools, let alone to publish the results.
Perhaps for these reasons, physiologists have been somewhat reluctant to embrace the new sensors. But now the field is mov-ing to close the gaps. New FRET pairs bring additional colors, and innovative concepts like circularly permuted fluorescent proteins are boosting sensor signals9,10. Studies such as that of Lohse and colleagues contribute to sensor validation, and the side-by-side comparison with previously published con-structs shows that, at least for cyclic nucleo-tides, we now do have a choice with respect to affinity and other properties. With the current equipment ready for these sensors, we can expect exciting biology to follow.
1. Nikolaev, V.O., Gambaryan, S. & Lohse, M.J. Nat. Methods 3, 23-25 (2006).
2. Honda, A. et al. Proc. Natl. Acad. Sci. USA 98, 2437–2442 (2001).
3. Sato, M., Hida, N., Ozawa, T. & Umezawa, Y. Anal. Chem. 72, 5918–5924 (2000).
4. Berridge, M.J., Bootman, M.D. & Roderick, H.L. Nat. Rev. Mol. Cell Biol. 4, 517–529 (2003).
5. De Koninck, P. & Schulman, H. Science 279, 227–230 (1998).
6. Tomida, T., Hirose, K., Takizawa, A., Shibasaki, F. & Iino, M. EMBO J. 22, 3825–3832 (2003).
7. Landa, L.R., Jr. et al. J. Biol. Chem. 280, 31294–31302 (2005).
8. Violin, J.D., Zhang, J., Tsien, R.Y. & Newton, A.C. J. Cell Biol. 161, 899–909 (2003).
9. Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).
10. Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Proc. Natl. Acad. Sci. USA 101, 10554–10559 (2004).
Kat
ie R
is
©20
06 N
atur
e P
ublis
hing
Gro
up
http
://w
ww
.nat
ure.
com
/nat
urem
eth
od
s
