FRET for studying intracellular signalling

MISCELLANEA

. FRET for studying intracellular s,gnalhng

FRETfor studying cAMPdependent protein klnase

Roger Y. Tsien, Brian J. Bacskai and Stephen R. Adams Fluorescence resonance energy transfer (FRET) is a quantum mechanical effect 1 that occurs when an excited fluorescent group and a light-absorbing group are located within a few nanometres of each other. If the energy of the excited fluorophore coincides roughly with the energy needed to excite the absorber, and if the distance and mutual orientations are suitable, then the energy can be transferred directly from the fluorophore (donor) to the absorber (acceptor) (Box 1). Measurement of the efficiency of FRET has been utilized in biochemistry as a spectroscopic 'ruler' to estimate the distance between donors and acceptors within a molecule2, 3. More recently, FRET has been applied to problems in cell biology: when donors and acceptors are contained within molecules that can form welldefined complexes or can diffuse into close proximity, FRET can show their degree of association. Here, we focus on the use of FRET for studying signalling events in cells. The special advantages of FRET for cell biologists are that it is nondestructive, sensitive to intermolecular distances of a few nanometres (far below the usual spatial resolution of light microscopy), and observable by fluorescence microscopy in intact cells and tissues, with high spatial and temporal resolution. The main limitation is the need to label the molecules specifically with suitable RogerTsienand donors and acceptors and to Brian Bacskaiare incorporate them back into living at the Department cells. of Pharmacology and Howard Detection of FRET HughesMedical FRET is manifested by three main Institute 0647; effects: reduction in the fluorand Stephen escence intensity of the donor fluAdams is at the orophore, reduction in the lifetime Departmentof of its excited state, and (if the acPharmacology, ceptor chromophore is itself fluorUniversityof escent) re-emission at the longer California, wavelengths (lower energies) San Diego, characteristic of the latter. The loss La Jolla,CA of donor brightness is the easiest 92093-0647, USA. effect to measure but it is also the 242

most vulnerable to confounding artifacts such as bleaching or other physical loss of donor fluorophores. An elegant method for normalizing such effects involves exhaustive photobleaching of the sample wh!!e integrating the fluorescence output4, s but this procedure is inherently destructive and prohibits further observation. Direct measurement of the nanosecond excited-state lifetimes is a sophisticated way to detect FRET, but the equipment is quite expensive for measurement in cuvettes and is not even commercially available for imaging by fluorescence microscopy, although the first setups are now being pioneered 6. We currently prefer to monitor FRET by using a second fluorophore as the acceptor and measuring the ratio of emissions from the donor and acceptor while exciting the donorT,8. Typically the donor is fluorescein and the acceptor is rhodarnine, although bodipy, cyanines, eosin and Texas Red are also worth considering 9. The ratioing method is very well adapted to laser-scanning confocal microscopy because the sample is excited with the blue-green 488 nm argon ion laser while the green and red emissions are separated with a dichroic mirror and monitored simultaneously. Therefore, high temporal and spatial resolution are maintained while variations in the number of fluorescein-rhodamine pairs are cancelled out. Photobleaching affects predominantly the fluorescein because it is the direct target of the 488 nm excitation and because it is generally more photolabile than rhodamine. Such photobleaching is partly but not completely cancelled by ratioing; loss of fluorescein decreases the fluorescein and sensitizedrhodamine emissions, but not the component of the rhodamine emission that comes froh-n direct excitation at 488 nm of the shortwavelength tail of the rhodamine absorbance band.

© 1993ElsevierSciencePublishersLtd (UK)0962-8924/93/$06.00

The use of FRET to reveal the extent of association of two molecules that form a well-defined complex or that can diffuse into close proximity has found wide application in cell biology. Classic examples4,S,lo are to track the mixing of fluorescently tagged lipids or membrane glycoproteins labelled with fluorescent lectins. A more recent application7, 8, with many implications for signal transduction, is to monitor cyclic AMP (cAMP).dependent protein kinase subunit dissociation. This kinase 11 is the only sensor of cAMP in most eukaryotic cells and is unusual among kinases in that its regulatory and catalytic subunits dissociate completely when the former binds cAMP. Measurement by FRET of this dissociation has thus provided the first method for continuous optical sensing of local intracellular cAMP concentrations and the extent of kinase activation in intact cells. Currently we prefer to attach fluorescein via an isothiocyanate group to the catalytic subunit and to attach tetramethylrhodamine onto the type II regulatory subunit via an N.hydroxysuccinimide linkage, both under conditions that favour attachment to lysines. The resulting protein is called FICRhR", short for fluorescein-labelled catalytic, rhodaminelabelled regulatory type II. When propedy labelled holoenzymeT, s is excited at fluorescein wavelengths, the ratio between fluorescein and rhodamine emissions increases by up to twofold when cAMP induces the dissociation of the subunits and abolishes the FRET from the fluorescein to the rhodamine (Fig. 1a). The dependence of the dissociation on the concentration of free cAMP is quite similar for the labelled and unlabelled holoenzyme (Fig. lb). Therefore FICRhR" has nearly the optimal affinity for cAMP, since the physiological concentrations of interest are precisely those that affect the native enzyme. Moreover, the kinase activity of the C subunit is unaffected by labelling~, suggesting that cAMP molecules that bind to exogenous FICRhR can still cause phosphorylations as if they had bound to endogenous

TRENDS IN CELLBIOLOGYVOL. 3 JULY1993

MISCELLANEA

kinase subunits. A sensor that was not an effective kinase would inherently tend to inhibit cAMP signalling by sequestering cAMP. Once the subunits have dissociated, their fluorescein and rhodamine tags permit the tracking of their separate fatesa. Although cAMP-dependent protein kinase is labelled under conditions that have been empirically adjusted to attach approximately one fluorescein and one rhodamine to the catalytic and regulatory subunit, respectively, we do not know at present which lysines are the targets. The use of FRET to obtain structural information about the kinase would require that known residues were specifically and homogeneously labelled° By contrast, measuring cAMP by FRET does not require such perfect protein chemistry because each batch can be empirically calibrated with known levels of cAMP as in Fig. lb. Obviously, similar calibrations should be possible using FRET to measure the percent formation of other macromolecular complexes, as long as the labels are close enough to give some rqET in the complex. FICRhR has been microinjected and imaged in a wide variety of systems (Refs 7,8,12,13; R. Civitelli, M. Mahaut-Smith, B. J. Bacskai, S. R. Adams, L. V. Avioli and R. Y. Tsien, unpublished; M. Hagiwara et el., unpublished). For example, in Aplysio sensory neurons13, confocal microscopy allowed separate observation of the nucleus, the surrounding cytoplasm of the cell body, and the peripheral processes. Bath application of the relevant neurotransmitter, 5-hydroxytryptamine, produced rapid increases in cAMP with remarkable spatial gradients (Fig. 2): cAMP rose to high concentrations in the processes, but was only slightly elevated in the cell body. These gradi. ents did not seem to be due to gross barriers to cAMP diffusion, since cAMP directly microinjected into the cell body was seen to spread into the processes by simpie diffusion with a diffusion constant just below 10-s cm2s-1, close to the value in free solution. Optical sections through the nucleus showed that it tended to exclude the holoen~me (injected

BOX 1 - QUANTITATIVE BASIS OF FLUORESCENCERESONANCE ENERGY TRANSFER

DONOR

ACCEPTOR

transition moment mo

transition moment mA

FRETis described in greater detail in reviews1,2,4,16,17and standard texts3,is on phot-~'~y~-i¢.~ -~.n_dfluorescence.The efficiency, E, of energy transfer is given by: E-

~+r 6

(1)

where r is the distance between the donor and accepter and Ro is the distance which energy transfer is 50% efficient. R0 is a complicated function of the spectral properties of the donor and accepter and of their mutual angular orientation, the formula for which is given by:

~o is the fluorescence quantum efficiency for the donor in the absence of the accepter. No is Avogadro'snumber, 6.023 x 1023mo1-1,n is the refractiveindex of the medium. The constants 9000 (In10)/(128~SN0) can be combined giving a value of 8.784 x 10-2s cm 3 mol 1-1. F~(~.) is the fluorescence intensity ef the donor b, teen wavelengths~. and ~.+d~ normalizedso that its integral over all wavelengths is 1. eA(~,)is the absorbance extinction coefficient (with dimensions of I mo1-1 cm-I) of the accepter at wavelength ~.. Therefore the integral in the above equation has overall dimensions of I mo1-1 cm 3 as needed to give R06the dimensions of cm6. K2 is a dimensionlessorientation factor: ~2 = (cosec- 3cosOocosO,)2

(3)

~2 is usually assumed to have a value of 2/3 because that is appropriate for dipoles that undergo random reorientation with respect to each other, for example if the donor and accepter are attached by flexible spacers. It can reach a maximum value of 4 if the donor and accepter dipoles and the vector connecting their centres are all parallel. K2 has a minimum value of 0 when the donor and accepter dipoles are perpendicular to each other and to the vector between them. Typical values of R0 are 2-6 nm, so that FRETmight be detectable up to about r= 10 nm. Because r is raised to the sixth power in Eqn 1, the energy transfer efficiency falls off quite strongly for r > R0, eventually as the inverse sixth power of r. FRET is by no means the only way that nearby chromophores can interact. Many other modes of energy transfer or electron transfer can come into play when r is less than 1-2 nm (Ref. 18). These interactions are quantitatively less predictable, so that such close approaches need either to be prevented or to be spectroscopicallydistinguished. Also, a trivial form of long-range interaction can occur if a large number of accepter molecules lie between the excited donor fluorophores and the light detector 3. Then light physically emitted by the donors gets reabsorbed by the accepters acting as a coloured filter. Unlike Lr~ FRET, this trivial reabsorption is strongly dependent on the overall size and geometry of the specimen. It is rarely a problem in fluorescence microscopy because the specimensare thin, transparentand viewed in epifluorescencemode so that no regions containing unexcited moleculeslie between the illumination zone and the detector. However, trivial reabsorbance can be a problem in macroscopic cuvettes in which emission is viewed at right angles to excitation.

TRENDSIN CELLBIOLOGYVOL. 3 IULY 1993

243

MISCELLANEA

100 /51

1.8

~M CAMP

.~ 7s :ac~iV:tkinnasa

| E ¢D

50

1.4

----t /

c

ratio

!! "

- 1 0

1.2 g

~. 25

•

500

•

•

*

520

•

•

,

I

540

,

-

,

,

•

.

•

i

560 580 wavelength(rim)

,

.

,

,

•

600

-

•

620

0

O.01

0.1 1 free [cAMP](I~M)

10

1.0 100

FIGURIE 1

(a) Fluorescenceemissionspectra of FICRhRu, in zero and saturating [cAMP], showing the spectralchange due to subunit dissociationand disruption of FRETby cAMP.The medium contained 135 mM KCI, 5 mM MgCI2, 3 mM ATP, 10 mM MOPS at pH 7.3, 22°C. The emissionspectra (not corrected for photomultiplier or monochromator ch~iracteristics)were obtained with 495 nm excitation before and after addition of 50 pM CAMP. (b) Calibration of the emissionratio change of FICRhRII (right curve and vertical scale)and comparison with the activation of unlabelled type II kinase(left curve and scale), both as a function of free [CAMP] (log scale).The FICRhRII (3 pM) was in a medium intended to simulate marine invertebratecytoplasm, consisting of 250 mM KOH, 100 mM glutamic acid, -300 mM MOPSfree acid adjusted to pH 7.3, 25 mM NaCt, S mM McJCI2, 3 mM ATP and 5 mM 2.mercaptoethanol, at 22°C. The protein was confined in a microdialysiscapillary (150 pm diameter and 9 kDa molecular weight cutoff), which was clamped on the stageof the laser-scanningconfocal microscopearid superfusedwith the above buffer to which variousconcentrationsof CAMPwere added. The ratio of fluorescencesemitted at 500-.530 nm (fluoresceinband) to that at >560 nm (rhodamine band), relativeto the ratio at zero [cAMP], was measuredwith the sameoptics as used for the neuronal imaging in Fig. 2. This method allowed control of free [cAMP] at submicromolarconcentrations while keeping the protein concentration and optical configuration similar to that used in ceils.The phosphorylating activity of unlabelled kinasewas measured at 22°C by the coupled assayof Cook et al. 19 with 40 nM holoenzyme in the above buffer without 2.mercaptoethanol but with 1 mM ATP, 0.1 mM Kemptide substrata,1 mM phosphoenolpyruvate, 7 U/ml pyruvate kinase,0.2 mM NADH, >15 U/ml lactatedehydrogenase,with varying amounts of cAMP. Free[cAMP] was calculated from the total [cAMP] by assumingthat two moleculesmust bind to each regulatory subunit to releasea catalytic subunit. Curveswere fitted by nonlinear least.squaresto the Hill equation with an apparent Kd of 0.42 pM and a Hill coefficient of 0.9 for the emissionratio, and an apparent Kd of 0.115IJ.Mand a Hill coefficientof 1.0 for the kinaseactivation. Of the 2.6-fold discrepancybetween the activation constants, -1 A-fold is because the apparent Kd for a fluorescence ratio differs from the true Kd by an optical factorS. The rest may be due to different assayconditions or effects of labelling.

into the cytoplasm) as long as cAMP remained at basal levels. Prolonged elevation of cAMP and dissociation of the holoenzyme caused gradual translocation of the catalytic subunit into the nucleus over tens of minutes. Translocation was reversible when cAMP was removed. The peak cAMP level reached by a variety of stimulation conditions correlated fairly well with the subsequent extent of nuclear transiocation. The observed gradient reflects a high [cAMP] where it is most needed for short-term responses, at the distal processes where the presynaptic terminals would be in vivo. Only strong or repeated stimulations are able to raise cAMP in the cell body sufficiently to release the catalytic subunit to diffuse into the nucleus, 244

phosphorylate transcription factors and cause longer-term changes in gene expression13 A contrasting application of cAMP measurement by FRET comes from a recent study by Sammak et all2 on the control of microtubule-mediated aggregation and dispersion of pigment granules in fish melanocytes. Such motility is not only important for camouflage of the fish, but also provides one of the clearest examples of rapid signal transduction from plasma membrane receptors to the microtubule cytoskeleton. Sammak et oL found that ¢¢2-adrenergic stimulation elevated cytosolic Ca2+, measured with fura-2, and decreased free cAMP, monitored by FRET imaging of FICRhR. However, artificial generation of Caz+ elevation or suppression of hormone-

induced CaZ+ transients showed that such CaZ+changes were neither necessary nor sufficient for granule motility. By contrast, pharmacological stimulation or inhibition of the cAMP pathway by a variety of permeant cAMP analogues and microinjected peptides or proteins correlated well with pigment granule dispersal or aggregation, suggesting that cAMP is the dominant second messenger controlling this type of motility. This study demonstrates that FICRhR works even in cells containing significant amounts of pigment and can detect rapid agonist-generated decreases in cAMP. A further interesting demonstration involved deliberate injection of excess FICRhR to determine whether the effect would mimic high or low cAMP. The latter proved to be the case, showing

TRENDS IN CELLBIOLOGYVOL. 3 JULY1993

MISCELLANEA

i ? :;.

RGURE 2 Bath application of semtonin (S-HT) to a single cultured sensory neuron from Aplysio colifomico generates an intracellular gradient of [cAMP]. The FICRhRn was pressure microinjected into the neuron and imaged with an emission-ratioing laser-scanning confocal microscope. Pseudocolours from blue to magenta indicate

increasing free [cAMP] with an approximate micromolar calibration shown by the colour scale (far right). 5-HT doses of 1, 5, 25 and 1 O0 I~M were sequentially applied and interspersed with washout periods of 20-25 min. In the resting state, [cAMP] is quite low and fairly uniform. With increasing 5-HT doses, the more distal processes eventually reach levels of [cAMP] that saturate the kinase by causing full dissociation (magenta pseudocolours), whereas the cell body remains poorly responsive to the transmitter (blue pseudocolours). For further discussion of the [cAMP] gradients and experimental methods, see Ref. 13.

phosphate [tns(1,4,5)P3] cause marked conformational changes in their native sensors [cGMP-dependent protein kinase, protein kinase C and the Ins(1,4,$)P3 receptor, respectively]. Despite the absence Other applications and future of subunit dissociation in these prospects In principle, FRET measurements cases, fluorescent sensors might analogous to these studies with be engineered by putting the two FICRhR should be useful to moni- fluorescent tags on the domains of tor protein-protein and protein- the protein that undergo the nucleic-acid association/dissociation largest change in mutual proxiin a wide variety of systems. Inter- mity. The particular virtue of fluoractions involving receptors, G-pro- escence methodology is that it can tein subunits, tyrosine kinases, be applied in intact cells with high calmodulin, effector proteins, tran- spatial and temporal resolution scription factors and promoter while the cells are performing sequences are of tremendous im- physiological functions. portance in biological signal transduction. Energy transfer has been References demonstrated in vitro between 1 FORSTER,T. (1948) Annalen der Physik 6, labelled complementary nucleic 55-75 acid sequences14and between Fos 2 STRYER,L. (1978) Annu. Rev. Biochem. 47, and Jun transcription factors in 819-845 3 LAKOWICZ,J. R. (1983) Principles of their heterodimerlS. Other second Fluorescence Spectroscopy, messengers such as cGMP, diacylPlenum Press glycerol, and inositol (1,4,5) tris-

that the dominant effect of excess FICRhR in these cells is to buffer cAMP rather than sensitize them to cAMP.

TRENDS IN CELL BIOLOGY VOL. 3 JULY 1993

4 JOVIN,T. M. and ARNDT-JOVIN,D. I. (i989) in Cell Structure and Function by Microspectrufluorometry (Kohen, E. and Hirschberg,J. G., eds), pp. 99-117, AcademicPress 5 JOVIN,T. M. and ARNDT-JOVIN,D. J. (1989) Annu. Ray. Biophys. Bfophys. Chem. 18, 271-308 6 LAKOWICZ,J. R., SZMACINSKI,H., NOWACZYK,K. and JOHNSON,M. L. (1992) Cell Calcium 13,131-147 7 ADAMS,S. R., HAROOTUNIAN, A. T., BUECHLER,Y. J., TAYLOR,S. S. and TSIEN, R. Y. (1991) Nature 349, 694-697 8 ADAMS,5. R., BACSKAI,B. J., TAYLOR, S. S. and TSIEN, R. Y. in Fluorescent Probes for Biological Activity of living Cells - A Practical Guide (Mason, W. T. and Relf, G., eds),Academic Press(in press) 9 TSIEN,R. Y. and WAGGONER,A. (1990) in Handbook of Biological Confocol Miuoscopy (Pawley,J., ed.), pp. 169-178, Plenum Press 10 USTER,P. S. and PAGANO, R. E. (1986) I. Cell Bfol. 103, 1221-1234 11 TAYLOR,S. S., BUECHLER,J. A. and YONEMOTO,W. (1990) Annu. Ray. 8iochem. 59, 971-1005 12 SAMMAK,P. J., ADAMS, S. R., HAROOTUNIAN,A. 1".,SCHLIWA, M. and TSIEN, R. Y. (1992)/. CelI8iol. 117, 57-72 13 BACSKAI,B. I. et al. (1993) Science260, 222-226 14 CARDULLO,R. A., AGRAWAL,S., FLARES, C., ZAMECNIK,P. C. and WOLF, D. E. (1988) Proc. Natl Acad. Sci. USA SS, 8790-8794 15 PATEL,L., ABATE,C. and CURRAN,T. (1990) Nature 347, $ 72-575 16 DOS REMEDIOS,C. G., MIKI, M. and BARDEN,J. A. (1987) J. Muscle Res. Cell Motil. 8, 97-117 17 HERMAN,8. (1989)in Fluorescence Microscopy of Living Cells in Culture Part B, Method~ in Cell Biology VoL 30 (Taylor, D. L and Wang, Y. L., eds), pp. 219-243, AcademicPress 18 TURRO,N. J. (1978) in Modern Molecular Photochemistry, p. 296, Benjamin/ Cummings 19 COOK, P. F., NEVILLE,M. E., VRANA, K. E., HARTL,F. T. and ROSKOSKI,R., JR (1982) Biochemistry 21, 5794-5799

In the Techniques section next month, Didier Picard discusses steroid-binding domains for regulating the functions of heterologous proteins in cis. 245