nmeth

8/6/2019 nmeth

1/26

2011

NatureAmerica,Inc.Allrightsreserved.

Articles

nAture methods | ADVANCE ONLINE PUBLICATION |

mba p a a ag, v gp p,

vg a a . ty a f

y ba q a p ba

. h a -p paza

py a a avaag ba

q y g ba p a , vg a ga. t q

a v agg G-p ava, ag

aa a a a p, a

ba p. cvy, ay ab

pb -p paza py aay x.

Many molecular processes taking place in living cells can be visu-alized1,2 by using genetically encoded fluorescent probes (usuallyrelying on various fluorescent proteins) and techniques of f luores-cence microscopy, such as fluorescence resonance energy trans-fer (FRET), fluorescence lifetime imaging, fluorescence recoveryafter photobleaching or fluorescence anisotropy imaging3,4. While

developing a genetically encoded probe of cell membrane voltage,we decided to investigate whether anisotropic optical propertiesof fluorescent proteins could be used to observe molecular proc-esses involving membrane proteins.

Optical properties of most molecules are anisotropic. Forsingle-photon electronic absorption, the absorption propertiesof a molecule are characterized by a vector, called the transitiondipole moment (TDM). The rate of light absorption by a moleculeis proportional to the squared cosine of the angle between theelectric field vector (polarization) of the excitation beam andthe TDM vector of the molecule5. The TDM direction thereforerepresents, in the reference frame of the molecule, the directionof excitation light polarization with maximum absorption rate.

To our knowledge, the direction of only one fluorescent proteinsTDM has been determined6. Two-photon absorption is describedby an absorptivity tensor, and effects of molecular orientationare generally complex7. For some molecules (rod-like)8, how-ever, two-photon absorption rate is proportional to the cosineto the fourth power of the angle between the excitation light

t-p paza py va p a

Josef Lazar14, Alexey Bondar1,2, Stepan Timr5 & Stuart J Firestein4

polarization and a vector (which we here term two-photonpseudo-TDM) describing the molecular orientation. Little isknown about fluorescent-protein absorptivity tensors, but GFPhas been shown6,9, in vitro, to exhibit anisotropic two-photonabsorption. Light emission is characterized by another TDM

(often similar in orientation to the excitation TDM), with fluores-cence preferentially emitted in directions perpendicular to theemission TDM and polarized in a TDM-containing plane.

Anisotropic optical properties of molecules can be observedin orientationally biased molecular assemblies. The cell mem-brane can provide sufficient orientational bias to dye molecules10,but fluorescent protein optical properties, linker flexibility andlimited photon counts might prevent observing anisotropicproperties in fluorescent proteinlabeled membrane proteins.Molecular rotation between excitation and emission, differencesbetween excitation and emission TDMs, and depolarization byan objective lens can hamper observations of fluorescence polari-zation. They should, however, have little effect on observing

dependence of fluorescence intensity on direction of excitationlight polarization or linear dichroism (LD).

Here we show that LD can indeed be observed in manyfluorescent proteintagged membrane proteins by two-photonpolarization microscopy (2PPM). The 2PPM technique pro-

vides information on molecular orientation and can be used forsensitive monitoring and quantification of protein-proteininteractions and conformational changes. We illustrate its usesby monitoring G-protein activation and changes in intracellularcalcium concentration.

results

maaa g

To investigate the possibility of using anisotropic proper-ties of fluorescent proteins to observe cellular processes, wedeveloped a mathematical model based on geometrical optics(Supplementary Fig. 1). We modeled absorption11 of a sphericalcell (approximating an oocyte, a yeast cell, a protoplast or, crudely,a mammalian cell) and a cylindrical cell (a generic elongated cell,

1Laboratory of Cell Biology, Institute of Nanobiology and Structural Biology, Global Change Research Centre, Academy of Sciences of the Czech Republic, Nove Hrady,Czech Republic. 2Department of Systems Biology, Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic. 3Department of Biochemistryand Molecular Biology, Faculty of Sciences, University of South Bohemia, Ceske Budejovice, Czech Republic. 4Department of Biological Sciences, Columbia University,New York, New York, USA. 5Department of Physics, Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic.Correspondence should be addressed to J.L. ([email protected]).

Received 13 ApRil 2010; Accepted 17 MAy 2011; published online 3 July 2011; doi:10.1038/nMeth.1643
http://www.nature.com/doifinder/10.1038/nmeth.1643http://www.nature.com/doifinder/10.1038/nmeth.1643

8/6/2019 nmeth

2/26

2011


| ADVANCE ONLINE PUBLICATION | nAture methods

Articles

a neuronal projection or a small section of an irregularly shapedcell). We kept the TDMs of fluorophore moieties attached tothe cell membrane either at a fixed angle 0 with respect to thecell membrane or modeled them as a Gaussian distribution ofangles , with a mean of0 and an s.d. (), to consider proteindynamics, conformational flexibility and nanoscopic membraneroughness. Our models for single-photon (Fig. 1) and two-

photon (Supplementary Fig. 2) linearly polarized excitationshowed that 0 can have notable effects on both the appearanceof fluorescently labeled cells and on the amount of observed fluo-rescence (Fig. 1a,b). We visualized differences in absorption oflight with distinct polarizations, or linear dichroism, by coloringfluorescence generated with horizontal polarization (Fh) magentaand fluorescence generated with vertical polarization (Fv) green(Fig. 1c,d). Conveniently, the hue of a pixel then directlyexpressed the dichroic ratio, r(r= Fh/Fv). We used ras a measureof LD, owing to its simplicity, experimental accessibility with littleimage processing and closeness of the log(r) distribution to a normaldistribution (allowing facile statistical analysis). A deviation ofrfrom 1 (and log(r) from 0) signified presence of LD.

Our model showed (Fig. 1e) that LD should be observ-able under a variety of suboptimal circumstances (includingmembrane roughness and protein confor-mational flexibility). In fact, absence of LDshould be an exception, generally occur-ring only for 0 = 54.7 for single-photonand 52.0 for two-photon absorption(so-called magic angle of fluorescenceanisotropy) and for very disordered fluoro-phore orientations. Our model also showed(Fig. 1f) that even if the distribution ofTDM tilt angles is wide ( = 20), a 1change in 0 (the mean TDM tilt) should

cause a 24% change in r(12% changes in

both Fh and Fv, in opposite directions). Fluorescence of constructswith TDM close to perpendicular to the cell membrane shouldbe particularly sensitive to changes in 0. Results for two-photon excitation (Supplementary Fig. 2) were similar to thoseobtained for single-photon excitation, with more pronounced LDapparent (typically about two times higher rand r/r). However,reliability of our two-photon model is limited because of approxi-

mations that had to be made in virtually complete absence ofinformation on the nature of two-photon absorptivity tensors influorescent proteins.

In summary, our mathematical model predicts that LD shouldbe widespread among fluorescently tagged membrane proteins,and it should be observable by single-photon polarization micros-copy and 2PPM. Even small changes in orientation of the fluo-rescent moiety should lead to observable changes in LD. Owingto the monotonic relationship between rand 0, with knowledgeof fluorescent protein TDMs and other parameters, it shouldbe possible to use polarization microscopy to gain quantitativeinsights into membrane protein structure and function. Thus,polarization microscopy should allow observation of a range of

molecular processes taking place in living cells and rational designof sensitive optical probes of these processes.

8

6

e

4

2

0

0 30

0 ()

60 901/4

0

5

10

20

45

1/2

1

2

4

16

64

256

r

2

log2

(r)

f

0 30

0 ()

60 900

5

10

15

(r/r)/

0(%)

0

5

10

20

45

0

0.1

0.2

0.3

log2

(r)/0

a

b

c

d

Fg | Mathematical models. (a,b) Simulatedimages o a luorescently labeled spherical cell(a) and cylindrical cell (b) shown as projectionso a single-photon conocalz-dimensionstack, or luorophore tilt angle 0 values 0,22.5, 45, 67.5 and 90 (let to right).(,) Simulated images or a sphericalcell () and cylindrical cell () showingluorescence excited by horizontally andvertically polarized light (Fh and Fv) coloredmagenta and green, respectively. Nongraycolor (excess o magenta or green) indicates

presence o LD. Direction o polarization andcoloring o corresponding luorescence isindicated by double-headed arrows. Orientationo the luorophore with respect to the cellmembrane (tilt angle 0) is indicated by theschematics in bottom right corners o individualimages. () LD, expressed as r= Fh/Fv andlog2(r), as a unction o mean luorophoretilt angle 0, or dierent widths (describedby ) o distribution o, or the cylindricalcell in . () Fractional changes in dichroicratio (r/r) o the cylindrical cell in and upon a change in mean tilt angle 0 by 1,or a range o starting tilt angles 0and tilt angle .

8/6/2019 nmeth

3/26

2011



Articles

F p a v

To test whether anisotropic effects predicted by our mathematicalmodel could be observed, we carried out measurements in livingcells (Fig. 2) on a construct termed doubly lipidated enhancedGFP (dleGFP)12 and constructs derived from it. DleGFP consistsof GFP and two lipophilic cell membrane targeting tags, thought toanchor the fluorescent protein to the cel l membrane in an almost

fixed orientation, with the fluorophore (and the single-photonTDM) close to perpendicular to the cell membrane (Fig. 2a).

When we observed cells expressing dleGFP using a wide-fieldfluorescence microscope with a polarizer placed in the excita-tion path, we discerned only subtle differences between imagesacquired with different polarizations (data not shown). Whenwe used a single-photon laser-scanning confocal microscope, weobserved anisotropic effects consistent with our mathematicalmodel (Fig. 2bf). These effects, however, were not particularlypronounced: the maximum observed dichroic ratio, rmax = (Fh/Fv)max, was 2.5 (15 cells). In contrast, when we used two-photonexcitation (Supplementary Fig. 3) to observe dleGFP-expressingcells, the observed LD was notable (rmax > 15, 200 cells, Fig. 2gk,

Supplementary Fig. 4 and Supplementary Video 1), consistentwith the pseudo-TDM orientation close to perpendicular to thecell membrane. In all cases, we also observed weak polarizationof the emitted fluorescence (data not shown). To confirm that itwas indeed the fixed orientation of the fluorophore that causedthe apparent LD, we created and examined, using 2PPM, twoconstructs based on dleGFP: internally lipidated eGFP (ileGFP)and C-terminally lipidated eGFP (cleGFP) (Fig. 2lo). Each ofthese contructs contained only one of the two original membrane-targeting tags, which presumably would not be sufficient to maintaina fixed orientation of the GFP moiety. Whereas cleGFP absorptionappeared isotropic (rmax < 1.1, 25 cells), ileGFP showed distinctLD (rmax = 6, 32 cells; two-photon pseudo-TDM close to perpen-

dicular to the cell membrane). Thus, the anisotropic phenomena

observed in dleGFP were indeed due to the orientation of thefluorophore. Furthermore, the LD observed in ileGFP, withfluorophore orientation likely only partly restricted because ofmembrane linkage through a loop region, was consistent withour mathematical model predicting that LD should be observ-able even in such cases. Membrane attachment through a flexibleC terminus (in cleGFP) did not give rise to LD.

Our results indicate that the sectioning ability of single-photonconfocal and two-photon imaging is beneficial for observationsof LD. The observed difference between single-photon and two-photon LD is likely due to nonlinearity of the two-photon exci-tation process (suppressing excitation by sides of the focal areacontaining unwanted polarizations), lower sensitivity of the two-photon polarization setup to minor alignment errors and stricterorientational requirements of two-photon excitation. Differentorientations of the single-photon TDM and two-photon pseudo-TDM in the eGFP molecule are also a possibility. Thus, likely fora combination of reasons, two-photon polarization fluorescencemicroscopy with non-descanned detection appears to reportfluorescent protein LD with markedly higher sensitivity than single-

photon microscopy. Our observations of LD in living mammaliancells using one-photon polarization microscopy and better using2PPM validate our mathematical model.

iagg p-p a: G-p px

To test whether 2PPM of fluorescently labeled membrane pro-teins can report on protein-protein interactions in living cells,we focused on processes involving heterotrimeric G proteins(composed of G, G and G subunits). Because there arefunctional, fluorescent proteinlabeled constructs available, weperformed most of our experiments on fluorescent proteintagged G subunits, particularly of the Gi-Go family. Weinvestigated 14 different Gi and Go constructs (Fig. 3 and

Supplementary Table 1).

DleGFP

CleGFPIleGFP

r

2.5

1

2.5

r

1.5

1

1.5

r

6

1

6

r

10

1

10

a

b c d e f

g h i j k

l m n o

Fg | Proo o principle. (a) Schematico the dleGFP construct. (b) Single-photonconocal images o a dleGFP-expressing cell.Direction o polarization and coloring ocorresponding luorescence is indicated as inFg . Shown are projections oz-dimensionstacks acquired with excitation light polarizedhorizontally (b) and vertically (); a compositeo images in b and colored magenta and green,respectively, without any color lookup table(LUT) adjustment (); a single conocal sliceo the same cell (); and the same image as

in , but ater application o an LUT suitableor displaying the range o dichroic ratio rinthe image (12.5; pixels exceeding this rangeappear pure magenta or pure green; only a smallnumber o such pixels are visible, indicating thatrmax = (Fh/Fv)max = ~2.5) (). (g) Images as inbbut acquired using two-photon excitation.() Schematic o the ileGFP construct. () Atwo-photon section o an ileGFP-expressing cell,processed as in but with a color scale coveringa narrower range o values as indicated.() Schematic o the cleGFP construct. () Atwo-photon section o a cleGFP-expressing cell,processed as in but with a dierent color scaleas indicated. All scale bars, 5 m.

8/6/2019 nmeth

4/26

2011



Articles

On the basis of LD, we could distinguish three distinctcategories of fluorescent proteinlabeled Gi-Go constructs.Constructs were named either as G subunit name followedby a fluorescent protein insertion site location and proteinname or as N-terminal tag name (GAP43) followed by a fluores-cent protein name and G subunit name. Two constructs (Gi2-Leu91-YFP13 and Go-Gly92-CFP

14) did not exhibit LD (rmax < 1.1)when overexpressed alone or when expressed together withG1 and G2. Another four constructs (Gi1-Leu91-CFP

15,Gi1-Leu91-YFP

16, Gi3-Leu91-YFP13 and Gi1-Ala114-YFP

17)showed pronounced LD (rmax = 26) both when expressed

alone and when expressed together with G1 and G2. A thirdgroup of constructs (GAP43-CFP-Gi1 (ref. 18), GAP43-CFP-Gi2 (ref. 18), GAP43-CFP-Gi3 (ref. 18) and Go-Leu91-YFP13) showed little or no LD when overexpressed alone butdistinct LD (rmax = 1.53) when expressed together with G1and G2.

These results illustrate structural differ-ences between the investigated constructs,even between constructs of similar overallamino acid sequences (Gi1-Leu91-YFP

16,Gi2-Leu91-YFP

13 and Go-Leu91-YFP13).

These structural differences are consist-ent with amino acid sequence diversity in

vicinity of the insertion site (the a-b loopregion) as well as with known19 functionaldifferences within this group of G subu-nits. Our results also support existenceof a physical interaction between at leastfour of the overexpressed fluorescent pro-teintagged G constructs and the overex-pressed G and Gsubunits (presumablyforming a Gcomplex), and suggest that2PPM can be used to monitor protein-protein interactions in live cells.

To ascertain whether the observedinteractions were physiological and not

an artifact owing to presence of multipleproteins overexpressed at high levels, we

carried out a series of G-protein activation experiments. Weexpressed four constructs together: a Gfluorescent protein,G1, G2 and a suitable G proteincoupled receptor (GPCR; typi-cally, the 2a adrenergic receptor tagged with YFP or CFP). Wethen activated the overexpressed receptor with an agonist (nore-pinephrine). Little or no changes in LD upon receptor activationcould be seen in G constructs lacking LD and in constructsshowing high LD both in absence and presence of G(data notshown). In contrast, all four Gfluorescent protein constructsthat showed differing amounts of LD in absence and in presenceof overexpressed G exhibited changes in LD upon receptor

activation (>10 cells examined for each construct, >80% cellsshowing responses; Fig. 4, Supplementary Videos 2 and 3and data not shown). Changes in LD in fluorescent proteintagged subunits could only be observed in cells in which expres-sion of a receptor (fluorescently labeled) was detectable. TheGo-Leu91-YFP

13 construct showed complete disappearance

r

1.5

1

1.5

r

1.5

1

1.5

r

1.5

1

1.5

r

5

1

5

r

1.5

1

1.5

r

1.5

1

1.5

r

1.5

1

1.5

r

5

1

5

a b c d

e f g h

Fg | 2PPM imaging o G-protein complexes. (a) Images o cells expressing luorescentlytagged G subunits GAP43-CFP-Gi2 (a), Gi2-Leu91-YFP (b), Go-Leu91-YFP () and Gi1-Leu91-YFP (). () Images o the same G subunits as in a but expressed together with G1 and G2.Coloring is as in Fg . Scale bars, 5 m.

Fg | 2PPM imaging o G-proteinactivation. (a) Cyan luorescence o acell expressing GAP43-CFP-Gi2, G1,G2 and 2a-adrenergic receptor-YFPbeore addition o norepinephrine (let),ater addition o norepinephrine (center)

and ater removal o norepinephrine(right). Coloring is as in Fg a .(b) Plot o LD (expressed as rand log2(r)) othe GAP43-CFP-Gi2expressing cell in a, as aunction o time. Triangles and squares denotedata rom the indicated horizontally andvertically oriented sections o the membrane,respectively. Dashed traces indicate s.e.m.,n = 110160 pixels. The 10-s period opresence o norepinephrine is indicated bya bar (top let). () Yellow luorescence o acell expressing Go-Leu91-YFP, G1, G2 and2aadrenergic receptorCFP beore additiono norepinephrine (let), ater addition o norepinephrine (center) and ater removal o norepinephrine (right). () Plot as in b but or theGo-Leu91-YFPexpressing cell in (n = 90160 pixels). All scale bars, 5 m.

r

2

1

2

r

1.5

1

1.5

0.50

0.25

0

0.25Log2

(r)

0.500 50

Time (s)

100 1501/1.4

1/1.2

r

r

1

1.2

1.4

a1

0.5

0

0.5Log2

(r)

10 50

Time (s)

100 1501/2

1/1.5

1

1.5

2

b

dc

HorizontalVertical

8/6/2019 nmeth

5/26

2011



Articles

of LD, consistent with dissociation of the G-G-Gcomplex.In contrast, the fluorescent proteintagged Gi subunits typicallyshowed only a decrease, not complete disappearance of LDupon activation, consistent either with incomplete (~80%)activation (Supplementary Note) or with rearrangement16(rather than dissociation17,20) of the G-protein complex uponactivation. Notably, a constitutively active mutant21 (Q204L) ofGi1-Leu91-YFP

16 showed significantly higher LD (P< 0.001)when expressed together with G and G(rmax = 2.8, 0 > 52,25 cells) than when expressed alone (rmax = 2.0, 0 > 52, 30 cells),providing strong evidence for existence of a G-protein trimer inactivated Gi subunits.

Our results show that it is possible to use 2PPM to visualizephysical interactions between subunits of heterotrimeric G proteins.The observed molecular interactions are physiological: that is,in presence of a suitable GPCR (and only in its presence) theyrespond to presence of a receptor agonist, in a fashion consistentwith current knowledge. The size of the responses observed duringG-protein activation is remarkable:F/Fof up to 50% in a particularchannel. In comparison, only ~2% changes in donor fluorescenceand 10% in acceptor fluorescence have been reported16 for FRETbetween Gfluorescent protein and Gfluorescent protein sub-units. Sensitivity of 2PPM is such that it should allow distinguish-ing between resting and activated states of the current Gi-Goconstructs within 200 s (Supplementary Discussion). Thus,

2PPM permits visualization of protein-protein interactions in liv-ing cells, yielding insights into molecular mechanisms of G-proteinactivation and allowing monitoring the process of activation ofGPCRs in live cells, in real time, with very high sensitivity.

iagg ag p a: a agg

To test the ability of 2PPM to report conformational changes inmembrane proteins, we used 2PPM to image changes in intracel-lular calcium concentration. Several lines of genetically encodedcalcium indicators have become commonly used22. Two of them(Cameleon-based23 and troponin-based24) rely on conformationalchanges reported by FRET. One of the cameleon sensors, lynD-3cpV25, is membrane-tethered. It consists of a calcium-sensing

domain sandwiched between two fluorescent proteins (CFP andcircular permuted (cp)Venus). About 30% changes in both donorand acceptor fluorescence have been reported for lynD3cpV uponan intracellular calcium concentration increase25.

Our 2PPM lynD3cpV observations (Fig. 5 and SupplementaryTable 2) showed that at low calcium concentrations, the CFPmoiety is in a fairly well-defined orientation with respect tothe cell membrane (rmax = 1.5; Fig. 5a). In contrast, the cpVenus moiety showed little LD (rmax = 1.07) in resting state cells.Upon an increase in intracellular calcium concentration throughstimulation by ATP, LD in cpVenus increased (rmax = 1.2).Upon removal of ATP, cpVenus LD returned to original values

(Fig. 5b,c and Supplementary Video 4). LD of CFP remainedconstant (data not shown). Calibration of cpVenus LD bycontrolling intracellular calcium concentration allowed char-acterization of the construct (saturated cpVenus rmax = 1.42,dissociation constant (Kd) = 0.64 M, Hill coefficient of 3.0;Supplementary Note) and determination of calcium concen-trations during ATP stimulation (0.40.6 M in different cells;mean = 0.495 M; rmax = 1.151.20) (Fig. 5d). These valuesare in very good agreement with results we obtained by FRET(Kd = 0.71 M, Hill coefficient of 2.6, ATP-induced calciumconcentrations 0.40.6 M, with mean of 0.505 M), althoughthe confidence intervals for calcium concentration valuesdetermined in individual cells were considerably larger in

2PPM experiments than in FRET experiments (Fig. 5d andSupplementary Discussion).

Our results are consistent with the lynD3cpV design anddemonstrate that even small changes in LD can be reproduciblyobserved, quantified and used to infer both structural and func-tional information. During ATP stimulation, cpVenusrmax/rmax =~0.15, corresponding to F/F of 7% in both the Fh and Fvchannels, in opposite directions. Both Fh and Fv are measured

virtually simultaneously, so although photobleaching adverselyaffects both values, it has little effect on the Fh/Fv ratio. Even themodest observed response size should (lynD3cpV responserate permitting) allow 2PPM observations of calcium spikes of13 ms (Supplementary Discussion).

0.2

0.1

0

0.1

Log2(r)

0.2

0.6

0.4

0.2Log2(rmax

)

Log2(F

cpVenus/FCFP)

0

0.5

1.5

2.5

0 50

Time (s)

100 150 0.001 0.01 0.1

[Ca2+

] (M)

[Ca2+

](M)

1 10 100

1/1.1

r1

1.1

0 150

0

0.5

FRETLD

Time (s)

0.40.4

0.5

0.6

0.5 0.6

[Ca2+]FRET

(M)

[Ca

2+]2PPM(

M

)c d

r

1.3

1.3

1

r

1.15

1.15

1

a bFg | 2PPM imaging o intracellularcalcium concentration through conormationalchanges in the calcium sensor lynD3cpV.(a) CFP signal o lynD3cpV. (b) cpVenusluorescence beore application o ATP (let),during application o ATP (center) and aterATP removal (right). Coloring is as inFg .All scale bars, 5 m. () Plot o cpVenus LD

(expressed as rand log2(r)) o the outlinedsections (inset) o the cell shown in a and b,as a unction o time. Triangles and squaresdenote data rom the indicated horizontally andvertically oriented sections o the membrane,respectively. Dashed traces indicate s.e.m.,n = 160200 pixels. The 40-s period opresence o ATP is indicated by a bar. () LDo cpVenus and FRET o lynD3cpV as a unctiono intracellular calcium concentration. Errorbars, s.e.m.; n = 1530 cells. The curve shownis a prediction or intermediate values oKd (0.68 M) and Hill coeicient (2.8) obtained rom LD and FRET measurements. Top inset, intracellular calciumconcentrations or six typical cells stimulated by ATP, determined by FRET (horizontal axis) and 2PPM (vertical axis). Error bars, s.e.m., n = 60400 pixels with|| < 3 and n = 10,00013,000 pixels used or FRET measurements. Bottom inset, experiment in , interpreted in terms o intracellular calcium concentration.

8/6/2019 nmeth

6/26

2011



Articles

discussion

The 2PPM method is applicable to many protein targets andfluorescent proteins (Supplementary Tables 1 and 2) in vari-ous cell types and organisms (including Saccharomyces cerevisiae,Caenorhabditis elegans and Drosophila melanogaster;Supplementary Fig. 5). Apart from membrane proteins, themethod is also applicable to cytoskeletal proteins and poten-

tially even to cytoplasmic proteins (after anchoring to the cellmembrane or cytoskeleton, or after photoselection). The tech-nique can be very sensitive (potentially allowing observationsof cellular processes with sub-millisecond temporal resolution,Supplementary Discussion), allows multiplexing and is resilientto bleaching artifacts. As 2PPM requires only one fluorescenttag (unlike FRET), many existing fluorescent proteintaggedconstructs are likely to act as 2PPM probes (similarly to theconstructs used in this study). The information 2PPM provides(fluorophore orientation and its changes) is largely complemen-tary to that provided by other methods, such as FRET. The 2PPMapproach can be used in rational development of both 2PPM andFRET-based probes.

In a basic form, 2PPM can easily be implemented on anytwo-photon microscope using a simple polarization modula-tor (Supplementary Fig. 3b). Such a setup allows observing LDin constructs with rmax > ~1.5. More sophisticated equipment(Supplementary Fig. 3c) allows detection of LD in most fluores-cent proteintagged membrane proteins, and observation andmonitoring of even very rapid processes.

Unlike other techniques, 2PPM relies on the shape and orienta-tion of the observed cell. Thus, cells with long sections of outlineoriented horizontally or vertical ly are more suitable for measure-ments than others. We have not found this to be a substantialdrawback (Supplementary Fig. 4), and in the future it is likely tobe mitigated by advances in polarization modulation and software.

Quantification of 2PPM data does require accounting for cellshape, but our results show that our procedure (SupplementaryFig. 6) allows accurate quantification of biophysical properties(Fig. 5d and Supplementary Discussion).

In principle, 2PPM is also capable of providing quantitativeinformation about membrane-protein structure, includingdetermining the orientation, with respect to the cell mem-brane or cytoskeleton, of the fluorescent protein (or anotherfluorescent moiety) attached to the studied protein. Suchdeterminations are currently not possible because of ourlimited knowledge of the micro- and nanoscopic geometryand dynamics of the cell membrane of living cells, the detailedoptical parameters of our imaging system and the nature of

two-photon absorptivity tensors of fluorescent proteins. Untilthese parameters are determined, information presentedin Supplementary Figure 2 can serve as a crude guide tostructural interpretation of 2PPM results.

Owing to its conceptual and experimental simplicity, robust-ness, wide applicability, availability of probes and high valueof information it can provide, we believe that 2PPM couldbecome an important method for visualization and analysisof molecular processes in living cells. Many phenomena thatare at present out of reach of imaging techniques, such as theinvestigation of G proteinGPCR interactions and observingindividual action potentials in neurons, may soon becomeobservable using 2PPM.

methods

Methods and any associated references are available in the onlineversion of the paper at http://www.nature.com/naturemethods/.

Note: Supplementary information is available on the Nature Methods website.

AcknowledGments

We thank L. Nedbal, Z. Benedikty, R. Uhl, M. Buenemann, Z. Peterlin andJ. Leps or discussions; C. Seebacher and A. Reshak or assistance withimaging; T. Bergmann, K. Tosnerova and members o the Institute o PhysicalBiology cell culture acility or technical assistance; R. Axel or inspiration;and G. Miesenboeck (Oxord University), M. Buenemann (Philipps UniversityMarburg), A. Tinker (University College London), R. Tsien (University oCaliornia, San Diego), M. Asahina-Jindrova (Institute o Parasitology,Academy o Sciences o the Czech Republic), C. Berlot (Geisinger Clinic),J. Blahos (Institute o Molecular Genetics, Academy o Sciences o the CzechRepublic), K. Deisseroth (Stanord University), S. Engelhardt (TechnicalUniversity Munich), N. Gautam (Washington University in St. Louis), A. Gilman(University o Texas, Dallas), S. Ikeda (US National Institute on Alcohol Abuseand Alcoholism), M. Jindra (Institute o Entomology, Academy o Scienceso the Czech Republic), T. Knopel (RIKEN Brain Science Institute), Y. Kubo(National Institute or Physiological Sciences, Japan), J. Ludwig (University oSouth Bohemia), R. Miller (Northwestern University), M. Rasenick (Universityo Illinois at Chicago), T. Montgomery, H. Sitte and T. Steinkellner (MedicalUniversity o Vienna) and M. Wildwater (Utrecht University) or constructs,

cells and animals. The research was supported by the European Commission(FP7 Marie Curie International Reintegration grant PIRG-GA-2007-209789MemSensors (J.L.), FP6-2005-Health project LSHG-CT-2007-037897Autoscreen (J.L.)), Columbia University Science Fellowship to J.L., McKnightInnovation in Neuroscience Award (S.J.F. and J.L.), Czech governmentinstitutional grants MSM6007665808, MSM6007665801 and AVOZ60870520(J.L.), EU Structural Funds grant CZ.1.07/2.3.00/09.0203 (J.L. and S.T.),University o South Bohemia ellowship (A.B.) and J.L.s personal savings.

Author contriButions

J.L. conceived the idea, carried out mathematical modeling and analyses,perormed initial microscopy experiments, developed image-processing sotware,directed the project and wrote the manuscript. A.B. perormed microscopyexperiments, prepared constructs, analyzed data and devised experimentalstrategies. S.T. developed sotware or quantitative analysis. S.J.F. contributedinspiration, consultations and unding.

comPetinG FinAnciAl interests

The authors declare competing inancial interests: details accompany theull-text HTML version o the paper at http://www.nature.com/naturemethods/.

Pb a p://.a./a/.

rp a p a avaab a p://.a.

/p/x..

1. Day, R.N. & Schauele, F. Fluorescent protein tools or studyingprotein dynamics in living cells: a review. J. Biomed. Opt., 031202(2008).

2. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing uorescentproteins. Nat. Methods, 905909 (2005).

3. Piston, D.W. & Rizzo, M.A. FRET by uorescence polarization microscopy.Methods Cell Biol.8, 415430 (2008).

4. Vrabioiu, A.M. & Mitchison, T.J. Structural insights into yeast septinorganization rom polarized uorescence microscopy. Nature, 466469(2006).

5. Lakowicz, J.R. Principles of Fluorescence Spectroscopy. 3 rd edn. (Springer,New York, 2006).

6. Shi, X. et al. Anomalous negative uorescence anisotropy in yellowuorescent protein (YFP 10C): quantitative analysis o FRET in YFP dimers.Biochemistry, 1440314417 (2007).

7. Callis, P.R. The theory o two-photon-induced uorescence anisotropy.in Topics in Fluorescence SpectroscopyVol. 5 (ed., Lakowicz, J.R.), 142(Plenum Press, New York, 1997).

8. Chen, S.Y. & Van Der Meer, B.W. Theory o two-photon induced anisotropydecay in membranes. Biophys. J., 15671575 (1993).

9. Volkmer, A., Subramaniam, V., Birch, D.J. & Jovin, T.M. One- and two-photon excited uorescence lietimes and anisotropy decays o greenuorescent proteins. Biophys. J.78, 15891598 (2000).
http://www.nature.com/naturemethods/http://www.nature.com/naturemethods/http://www.nature.com/naturemethods/http://www.nature.com/naturemethods/

8/6/2019 nmeth

7/26

2011


nAture methods | ADVANCE ONLINE PUBLICATION | 7

Articles

10. Benninger, R.K., Onelt, B., Neil, M.A., Davis, D.M. & French, P.M.Fluorescence imaging o two-photon linear dichroism: cholesterol depletiondisrupts molecular orientation in cell membranes. Biophys. J.88,609622 (2005).

11. Axelrod, D. Carbocyanine dye orientation in red cell membrane studiedby microscopic uorescence polarization. Biophys. J., 557573(1979).

12. Roorda, R.D., Hohl, T.M., Toledo-Crow, R. & Miesenbock, G. Video-ratenonlinear microscopy o neuronal membrane dynamics with genetically

encoded probes.J. Neurophysiol.9, 609621 (2004).13. Frank, M., Thumer, L., Lohse, M.J. & Bunemann, M. G Protein activation

without subunit dissociation depends on a G{alpha}(i)-specifc region.J. Biol. Chem.80, 2458424590 (2005).

14. Azpiazu, I. & Gautam, N. A uorescence resonance energy transer-based sensor indicates that receptor access to a G protein isunrestricted in a living mammalian cell. J. Biol. Chem.79,2770927718 (2004).

15. Hein, P., Frank, M., Homann, C., Lohse, M.J. & Bunemann, M. Dynamicso receptor/G protein coupling in living cells. EMBO J., 41064114(2005).

16. Bunemann, M., Frank, M. & Lohse, M.J. Gi protein activation in intactcells involves subunit rearrangement rather than dissociation. Proc. Natl.Acad. Sci. USA00, 1607716082 (2003).

17. Gibson, S.K. & Gilman, A.G. Gi and G subunits both defne selectivityo G protein activation by 2-adrenergic receptors. Proc. Natl. Acad.Sci. USA0, 212217 (2006).

18. Leaney, J.L., Benians, A., Graves, F.M. & Tinker, A. A novel strategy toengineer unctional uorescent inhibitory G-protein alpha subunits.J. Biol. Chem.77, 2880328809 (2002).

19. Foerster, K. et al. Cardioprotection specifc or the G protein Gi2 inchronic adrenergic signaling through 2-adrenoceptors. Proc. Natl. Acad.Sci. USA00, 1447514480 (2003).

20. Digby, G.J., Lober, R.M., Sethi, P.R. & Lambert, N.A. Some G proteinheterotrimers physically dissociate in living cells. Proc. Natl. Acad. Sci.USA0, 1778917794 (2006).

21. Kroll, S.D. et al. The Q205LGo-alpha subunit expressed in NIH-3T3 cellsinduces transormation.J. Biol. Chem.7, 2318323188 (1992).

22. Hendel, T. et al. Fluorescence changes o genetic calcium indicators andOGB-1 correlated with neural activity and calcium in vivo and in vitro.J. Neurosci.8, 73997411 (2008).

23. Miyawaki, A. et al. Fluorescent indicators or Ca2+ based on greenuorescent proteins and calmodulin. Nature88, 882887 (1997).

24. Mank, M. et al. A genetically encoded calcium indicator or chronicin vivo two-photon imaging. Nat. Methods, 805811 (2008).

25. Palmer, A.E. et al. Ca2+ indicators based on computationally redesignedcalmodulin-peptide pairs. Chem. Biol., 521530 (2006).

8/6/2019 nmeth

8/26

2011


doi:10.1038/nmeth.1643nAture methods

online methods

Mathematical modeling. We developed a mathematical model ofa spherical and a cylindrical cell (Supplementary Fig. 1) based ongeometrical optics and similar to published models10,11. Briefly,the surface of the cell contained idealized fluorophore molecules,each possessing a single absorption and emission TDM with iden-tical orientations. Angles and (spherical cell), or an angle

and ay coordinate (cylindrical cell) defined the position of afluorescent molecule on the surface of the model cell. We definedthe orientation of the TDM for each fluorophore molecule by anangle between the TDM and a normal to the cell membrane,and an angle describing rotation along an axis normal to thecell membrane. We approximated excitation polarization by asingle, linear polarization in the focal plane. The model took intoaccount collection of fluorescence by a high-numerical-aperturelens. We carried out modeling for a range of discreet values of,for Gaussian distributions ofdescribed by a mean value of0and an s.d., , and for other distributions. We performed somemodeling of individual confocal or two-photon slices, but we pre-ferred to model z-dimension stack projections, to avoid effects

of different cell sizes. For two-photon excitation, we assumed acosine to the fourth power relationship of the rate of absorptionon the angle between the electric field vector of the excitation lightand the two-photon pseudo-TDM. The models were implementedusing Mathematica and Perl. Images were generated using thePerlMagick Perl module.

Constructs.DleGFP12 was a gift from G. Miesenboeck. FromdleGFPwe derived the cleGFPand ileGFPconstructs throughremoval of one of the membrane targeting tags by PCR (Phusionpolymerase, New England Biolabs (NEB)) using suitable primers(cleGFP_Fand cleGFP_R for cleGFP, and ileGFP_Fand ileGFP_Rfor ileGFP; Supplementary Table 3). We purified PCR products

by agarose gel electrophoresis, extracted the DNA from the gel(QIAexII gel extraction kit, Qiagen), phosphorylated the DNA(T4 polynucleotide kinase, NEB), circularized it (T4 DNA ligase,NEB) and transformed it into Escherichia coli (DH5, Invitrogen),using standard or manufacturer-recommended procedures. Foreach construct, we grew two bacterial colonies in 5 ml of LBmedium with 100 mg l1 ampicillin. We isolated the plasmids(QIAquick Spin kit, Qiagen) and verified sequences of the insertsby DNA sequencing (Agowa).

Other constructs were gifts from M. Buenemann (Gi1-Leu91-YFP, Gi1-Leu91-CFP, Gi2-Leu91-YFP, Gi3-Leu91-YFP,Go-Leu91-YFP, Gs-Gly72-YFP, 2aAR-YFP, 2aAR-CFP,a2A adenosine receptorYFP, 2AR-CFP and 2AR-YFP),

A. Tinker (GAP43-CFP-Gi1, GAP43-CFP-Gi2, GAP43-CFP-Gi3, GAP43-CFP-Go and GAP43-YFP-Go), Y. Kubo(mGluR1-i1-YFP, mGluR1-i2-YFP and mGluR1-C-tail-YFP), R. Miller (G1-YFP), S. Ikeda (G1, G2 and G2-CFP),M. Rasenick (Gs-D71-GFP), A. Gilman (Gi1-Ala114-YFPand Gi2-Ala114-YFP), N. Gautam (Gi2-Leu91-CFPand Go-Gly92-CFP), C. Berlot (Gq-Phe124-GFP, Gs-Gly72-CFPandPKC-DsRed), J. Blahos (mGluR2-GFP), S. Engelhardt (1AR-Cer/YFP), T. Knopfel (VSFP 3.1, Addgene plasmid 18951),K. Deisseroth (opto-1-AR-YFPand opto-2-AR-YFP; Addgeneplasmids 20947 and 20948), E. Boyden (FCK-ChR2-GFP andFCK-Halo-GFP; Addgene plasmids 15814 and 14750, respec-tively) and R. Tsien (SuperGluSnFR and lynD3cpV).

Mammalian cell culture. We cultured HEK293 cells at 37 Cunder an atmosphere of 95% air, 5% CO2, in Dulbeccos modifiedEagles medium with Glutamax I and high glucose (Invitrogen),supplemented with 10% fetal bovine serum. Before observa-tion, we typically plated cells on 8-chamber microscopy slides(-Slides, Ibidi) and transfected them using Lipofectamine 2000(Invitrogen), according to the manufacturers protocol. We per-

formed microscopy experiments 2448 h after transfection. Weperformed G-protein activation and calcium-imaging experimentsin flow chambers (-Slide I0.8 Luer slides, Ibidi), using a peristalticpump (Minipuls3, Gilson). We washed cells with HEPES-bufferedHanks balanced salt solution (pH 7.4) and stimulated them withnorepinephrine (()-norepinephrine (+)-bitartrate salt; Sigma)at a final concentration of 1 M for G-protein activation, orwith ATP (Sigma) at a final concentration of 10 M for calciumimaging. To calibrate lynD3cpV responses, we applied calciumchlorideEGTA buffers containing ionomycin (Sigma, 5 M) and1 nM to 39 M of free calcium for 30 min before imaging.

Polarization fluorescence microscopy. Polarization microscopy

was performed on a customized laser-scanning confocal micro-scope iMic (Till Photonics) equipped with a Yanus beam scan-ner (Till Photonics), a 488-nm argon laser (LGK 7812-1, Zeiss)for single-photon confocal imaging and a tunable pulsed tita-nium:sapphire laser (Chameleon Ultra II with GVD compensa-tion, Coherent) operated at 800 nm (CFP) or 960 nm (GFP, YFPand DsRed) for two-photon imaging. We used a UApoPlan/IR60, numerical aperture (NA) 1.2 water-immersion objectivelens (Olympus). For single-photon confocal imaging, we used acombination of a long-pass dichroic beam splitter (FF495-Di02,Semrock) and a Brightline 500/24 (Semrock) emission filter. Fortwo-photon imaging (with non-descanned detection), we used along-pass dichroic (FF705-Di01, Semrock) and a suitable emis-

sion filter (Brightline 479/40 for CFP, Brightline 500/24 for GFPand Brightline 542/27 for YFP; all Semrock), combined with aninfrared-blocking filter (HQ700SP-2P, Chroma). Fluorescence wasdetected by a photomultiplier (R6357, Hamamatsu Photonics),operated at 700900 V, providing 16-bit output.

A polarization modulator (Supplementary Fig. 3) allowedrotating polarization of the excitation beam. In our initial experi-ments (Figs. 2 and 3, Supplementary Fig. 4 and SupplementaryVideo 1) we used a simple, manually operated polarization modu-lator (Supplementary Fig. 3b) consisting of a Glan-laser polar-izing beam splitter (CVI Laser) and a rotatable half-wave plate(488 nm zero-order, or 6901,080 nm achromatic; Thorlabs).We acquired, successively, two images of the same cell, with the

polarization of the excitation beam oriented horizontally and ver-tically in the reference frame of the acquired image. All imageswere acquired at 100200 nm pixel size (typically, 100 nm) and10 s pixel1 acquisition time.

In our later experiments (Figs. 45, Supplementary Figs. 56,and Supplementary Videos 24) we used a rapid polarizationmodulator (RPM) (Supplementary Fig. 3c) custom-made byBME Bergmann. The RPM consisted of a Pockels cell (RTP-3-20-AR800-1000, Leysop) and a high-voltage driver synchronized withthe microscope, so that polarization of the excitation beam wouldalternate (between horizontal and vertical) between acquisitionof subsequent pixels. Typically, we acquired an image at 50 nm 100 nm pixel size and 10 s pixel dwell time. We split the resulting

8/6/2019 nmeth

9/26

2011


doi:10.1038/nmeth.1643 nAture methods

image into two images, one consisting of odd-numbered pixels ofthe original image (acquired with horizontal polarization) and theother consisting of even-numbered pixels of the original image(vertical polarization). We imaged and quantitatively analyzed atleast 15 cells for each construct or combination of constructs.

Image processing. We performed basic image processing with

ImageJ, using standard ImageJ tools and in-housedevelopedmacros. We subtracted background of acquired images andadjusted brightness (but not contrast). We colored correspond-ing images acquired with horizontal and vertical polarizationmagenta (equal intensity blue and red) and green, respectively,and merged them into a composite image. When desired, theresulting image was colored using a color LUT designed to showa suitable range ofFh/Fv ratios while keeping the overall bright-ness constant. This procedure allowed simultaneous visualizationof LD (of any size) and total fluorescence intensity. For small

values of LD (r< 1.5), we applied a small (

8/6/2019 nmeth

10/26

Nature Methods

Two-photon polarization microscopy reveals protein structure

and function

Josef Lazar14, Alexey Bondar1,2, Stepan Timr5 & Stuart J Firestein4

Supplementary Figure 1 Mathematical models.

Supplementary Figure 2 Results of mathematical modelling for two-photon excitation.

Supplementary Figure 3 Setup for two-photon polarization microscopy.

Supplementary Figure 4 A 2PPM image of HEK293 cells expressing dleGFP.

Supplementary Figure 5 Examples of cells and constructs showing linear dichroism.

Supplementary Figure 6 Quantitation of linear dichroism.

Supplementary Table 1 G-protein constructs

Supplementary Table 2 NonG-protein constructs

Supplementary Table 3 Primers

Supplementary Note Linear dichroism of a two-state system.

Supplementary Discussion Signal-to-noise analysis.

Note: Supplementary Videos 14 are available on the Nature Methods website.

8/6/2019 nmeth

11/26

0

y

x

z

y

x

z

y

a b c

Supplementary Figure 1: Mathematical models.

(a) Model of a spherical cell. Position of the fluorophore on the surface of the cell is described

by angles and . (b) Model of a cylindrical cell. Position of the fluorophore on the surface

of the cell is described by coordinates and y. (c) Definition of variables describing the orienta-

tion of the fluorophore (transition dipole moment): mean tilt angle 0, standard deviation , and

rotational angle. (d) Behavior of a linearly polarized laser beam used for single-photon excita-

tion, passing through an objective lens (geometrical optics approximation). Polarization of the

beam remains perpendicular to the direction of light propagation. In the focal area, the present

directions of polarization add up in a vector fashion, restoring the original polarization of thelaser beam. Fluorescence excitation occurs throughout the light double cone created by the

objective lens. Observed area can be restricted by use of a confocal pinhole. (e) Behavior of a

linearly polarized laser beam used for two-photon excitation, passing through an objective lens

(geometrical optics approximation). Similar to d, but with excitation occurring invariably only in

the focal area.

Focal point:

Elsewhere:

=

Focal point:

Elsewhere:

=

d e

8/6/2019 nmeth

12/26

0

5

10

20

45

0 30 60 90

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0

10%

20%

30%

40%

0()

log2

(r)/0 (

r/r)/0

0

5

10

20

45

0 30 60 90-2

0

2

4

6

8

10

12

14

16

1/4

1

4

16

64

256

1024

4096

16384

0()

log2

(r)

r

dc

b

a

Supplementary Figure 2: Results of mathematical modeling for two-photon excitation.

(a) Simulated images of a fluorescently labeled spherical cell a projection of a z-stack, for

TDM tilt angle 0 values 0, 22.5, 45, 67.5, and 90. Direction of polarization and coloring of

corresponding fluorescence is indicated by double-headed arrows. Linear dichroism (non-gray

color) is present for all shown 0 values. (b) Same as in a, but for a cylindrical cell. (c) Graph

of observed LD expressed as r (r= Fh/F

v) and log

2(r), as a function of0 and for experimental

arrangement in b. LD (r 1) is generally present, except when 0 = 50-55 or is very high(45). (d) Graph of expected fractional changes in dichroic ratio (r/r) upon a change in mean

tilt angle 0, for experimental arrangement in b. The graph, in effect, shows the percentage

change in rupon a change in the mean tilt angle 0 by 1, for different starting values of0 (x-

axis) and for different standard deviations () of the Gaussian distribution of tilt angles. Even for

wide distributions of ( = 20), a 1 change in 0 typically leads to a sizeable (2-4%) change

in r.

8/6/2019 nmeth

13/26

laserpolarization

modulator

detector

scanning mirrors

dichroic mirror

objective lens

a

b c

Supplementary Figure 3: Setup for two-photon polarization microscopy.

(a) Schematic diagram of a two-photon polarization microscope. (b) A simple polarization

modulator composed of a Glan-laser polarization beamsplitter and a manually rotatable half-

wave plate. (c) A rapid polarization modulator (RPM) based on a Pockels cell, driven in syn-

chrony with scanning of the microscope.

8/6/2019 nmeth

14/26

Supplementary Figure 4: A 2PPM image of HEK293 cells expressing dleGFP.The image was created by merging images acquired with horizontal and vertical polarization

(colored magenta and green, respectively). Polarization of the excitation beam was rotated

manually between acquisition of individual images. No background subtraction, brightness

or contrast adjustments were applied. Scale bar: 10 m.

8/6/2019 nmeth

15/26

1.3

1.3

1

r

1.3

1.3

1

r

1.3

1.3

1

r

6

6

1

r

10

10

1

r

1.3

1.3

1

r

1.3

1.3

1

r

1.3

1.3

1

r

1.3

1.3

1

r

1.3

1.3

1

r

f g h i j

a b c d e

Supplementary Figure 5: Examples of cells and constructs showing linear dichroism.

Coloring as in Figs.2-5. Scale bars: 5 m. a-f, HEK293 cells. (a) Protein kinase C-DsRed (C-

terminal fusion)1. The top, non-activated cell shows cytoplasmic localization of fluorescence and

no LD, unlike the bottom, activated cell (rmax = 1.5, 0 < 52). (b) a2A-adenosine receptor-YFP (C-terminal fusion)2; rmax = 1.5, 0 > 52. (c) Metabotrapic glutamate receptor mGluR1-YFP

(intracellular loop 1 insertion)3; rmax = 1.5, 0 >> 52. (d) 2a-adrenergic receptor-CFP (C-terminal

fusion)4, rmax = 1.5, 0 > 52. (e) VSFP3.1, an engineered sensor of membrane voltage5, rmax =

1.5, 0 > 52. (f) Microtubule associated protein tau-GFP (C-terminal fusion)6. LD is visible both in

cytoplasmic and in membrane associated microtubules (rmax = 1.5, 0 > 52). (g) DleGFP7

expressed in a rat hippocampal neuron (rmax = 1.5, 0 < 52). (h) Yeast S. cerevisiae expressing

potassium channel TOK1-GFP (rmax = 1.5, 0 < 52)8. (i) An epithelial seam cell of a live C.

elegans worm, expressing pleckstrin-homology domain-GFP (rmax = 1.5, 0 < 52). (j) Epithelium

of fruitfly D. melanogasterexpressing E-cadherin-GFP (rmax = 1.5, 0 > 52)9.

8/6/2019 nmeth

16/26

db

a

r

5

3

1

1-1 0 0.5-0.5 1.5-1.5

r

0

3

2

1

01

2

-2-1

-2-1

01

20

4

0

/2

-/2

- /2 0/2

0

/2

-/2

c

Supplementary Figure 6: Quantitation of linear dichroism.

(a) Analysis of geometry of an ileGFP expressing cell, a single optical section. From left to

right: an image used for construct characterization, processed as in Fig. 2; pixels judged to be

part of the cell outline; a spline approximating the cell outline; the same spline, colored accord-

ing to orientation (angle ); pixels of the cell outline colored according to the orientation (angle

) of the closest point on the spline, with color bar shown. (b) Graph of LD as a function of cell

outline orientation (LD/CSO relationship), for the cell in a. The data was fitted by a curvepredicted for a particular combination of fluorophore tilt angle 0 and tilt angle distribution

width (0 = 0.7, = 0.1). (c) Three-dimensional reconstruction of an ileGFP expressing cell,

shown as a triangular mesh, and colored according to cell surface orientation (angles , ),

with color bar shown. (d) Graph of LD as a function of cell surface orientation, for the cell in

c. The data was fitted by a surface predicted for the same combination of0 and as in b.

8/6/2019 nmeth

17/26

Supplementary Table 1: G-protein constructs

When expressed aloneWhen co-expressed

with G, GG-protein construct

Design /

fluorescent

proteininsertion site

LD rmax 0 n LD rmax 0 n

GAP43-CFP-Gi11020-AA tag

CFP Gi1- < 1.05 N/A 20 + 2.0 < 52 25

Gi1-Leu91-YFP11 91-92 + 2.8 > 52 38 + 3.1 > 52 33

Q204L-Gi1-Leu91-YFP11

91-92 + 2.1 > 52 25 + 2.9 > 52 30

Gi1-Leu91-CFP12 91-92 + 2.0 > 52 24 + 3.1 > 52 20

Gi1-Ala114-YFP13

114-115 + 2.1 > 52 30 + 3.2 > 52 30


CFP Gi2- < 1.05 N/A 35 + 2.1 < 52 37

Gi2-Leu91-YFP14 91-92 - < 1.05 N/A 30 - < 1.05 N/A 25

Gi2-Ala114- YFP13 114-115 + 1.1 > 52 16 + 1.1 > 52 15


CFP Gi3- < 1.05 N/A 22 + 2.1 < 52 18

Gi3-Leu91-YFP14 91-92 + 2.0 > 52 17 + 3.2 > 52 18

GAP43-CFP-Go10 20-AA tag CFP Go - < 1.05 N/A 15 + 1.6 < 52 15

GAP43-YFP-Go1020-AA tag

YFP Go- < 1.05 N/A 20 + 1.6 < 52 22

Go-Leu91-YFP14 91-92 - < 1.05 N/A 52 + 1.7 < 52 62

Go-Gly92-CFP15 92-93 - < 1.05 N/A 15 - < 1.05 N/A 15

Gs-Asp71-GFP16 71-82 - < 1.1 N/A 15 + 1.3 < 52 15

Gs-Gly72-CFP17 72-85 - < 1.05 N/A 20 + 1.8 < 52 25

Gs-Gly72-YFP2 72-85 - < 1.1 N/A 15 - < 1.1 N/A 15

Gq-Phe124-GFP1 124-125 + 1.8 < 52 25 + 1.8 < 52 25

G1-YFP18 N-terminal - < 1.05 N/A 15 + 1.4 > 52 20

8/6/2019 nmeth

18/26

Supplementary Table 2: Non-G-protein constructs

Construct LD rmax 0 n

2aAR-CFP4 + 1.7 < 52 35

2aAR-YFP4 + 1.3 < 52 30

2AR-YFP4 -

8/6/2019 nmeth

19/26

Supplementary Table 3: Primers

Primer name Sequence

cleGFP_F TTTAATCTGTGTTGTAACTC

cleGFP_R GATGGAGGCGTTCAACTAG

ileGFP_F ACGACCCTAATGTGTACCGATTCT

ileGFP_R CCGCTTCCCTTTAGTGAG

8/6/2019 nmeth

20/26

SUPPLEMENTARY NOTE

LD of a two-state system

The composition of a mixture (AB) of two states (A, B) can be inferred from the dichroic

ratio of the mixture (rAB), if the dichroic ratios of the individual components (rA, rB) of

the mixture are known. IfxA,xB are the fractions of the protein present in the form A and

B, respectively, and we assume that the total amount of fluorescence (F= FA + FB) is

approximately independent ofxA andxB (FA =xA . F; FB =xB . F), we can write:

vA

AF

r hAF

, andvB

BF

r hBF

. (Eq. 1, 2)

Thus,AAA

vArrr

F vAAvAAhAFFxFFF

, (Eq. 3)

and therefore Fr

FA

vA1

xA (Eq. 4)

Similarly, it can be shown that

Fr

FB

vB1

xA1 , F

rF

A

hA1

xr AA , and F

rF

B

hB1

xr AB )1( . (Eq. 5, 6, 7)

Thus, the dichroic ratio of a two-component system is:

)1)(1()1()1)(1()1(

1

)1(

1

11

AABA

BAABAA

B

A

A

A

BA

vBvA

hBhAAB

rxrxrrxrrx

Fr

xF

r

x

F

r

F

rFFFFr

)1( BAAA rxrx

(Eq. 8)

)(1 BAAAAB

rrxrr

)()1( BAAAB rrxrr (Eq. 9)

8/6/2019 nmeth

21/26

)(1 BAAAAB

rrxrr

)()()()1( BAABAABBAABAB rrxrrxrrrxrrr (Eq.10)

)(1)(1 BAAAB

BAAA

BABrrxr

rrrxr

rr

)1)(()()( BBAABAABAAB rrrxrrxrrxr

(Eq.11)

The fraction of the component A can then be determined:

)1)(( )1)((

ABBA

Arrr

x

ABAB rrr

)(BAABAB rrxrr

(Eq. 12)

For values ofrA and rB similar to each other [specifically, for (rA-rB) considerably smaller

than (1+r

A)], Eq. 11 can be approximated by a simple linear relationship,

(Eq. 13)

and the fraction of component A then is:

)(

)(

BA

BABA

rr

rrx

(Eq. 14)

Apart from determinations of composition of mixtures, the above equations can

also be used for determining Kd, by substituting forxA in Eq. 12 from a suitable

equilibrium equation [such as the Hill equation we used for determining the Kd of

lynD3cpV,xA = [Ca2+

]n

/ (Kdn

+ [Ca2+

]n) ]. Alternatively, Kd values can be determined

by applying a standard mathematical description developed for ratiometric FRET

imaging24

to ratiometric 2PPM data (Fh/Fv). For a first order equilibrium reaction we can

write:

[Ca2+

]

vA

vB

ABA

BABd

FrrK

Frr(Eq. 15)

The term FvB/FvA is not easily experimentally accessible. However, if we assume (as we

8/6/2019 nmeth

22/26

did in Eqs. 1-14) that the total fluorescence intensity (Fh + Fv) is the same for forms A

and B, we can approximate FvB/FvA by (rA + 1)/(rB + 1). For a system described by a Hill

equation we can then write:

[Ca2+

]n

1

)(B

A

ABA

BABn

drrr

K 1rrr

(Eq. 16)

Eq. 16 is equivalent to a combination of Eq. 12 and the Hill equation.

8/6/2019 nmeth

23/26

SUPPLEMENTARY DISCUSSION

Signal-to-noise analysis

Calibration of photomultiplier detector response through Poisson noise analysis

has allowed us to interpret pixel intensities in terms of photon counts. Typically, during

the pixel dwell time (10 s) used by our setup, 100-200 photons were detected in each

pixel within the cell outline. The 95% confidence interval (95%CI) for a single

measurement and Poisson distribution of 150 events is 127-177. Thus, a single pixel

allows measurement of fluorescence intensity with ~20% accuracy. A Poisson

distribution of the mean value of 150, being far from 0, can be approximated by a normal

distribution with a mean of 150 and variance of 150 (thus, with both the standard

deviation and standard error equal to 150 = ~12). For values ofr= Fh/Fv close to 1 we

can estimate the standard deviation ofrto be 2 standard deviation of individual

fluorescence measurement/mean fluorescence intensity = 1.41 12 / 150 = 0.11.

Therefore, from a single pixel we know the value ofrwithin ~22%, with 95% confidence.

A short stretch (0.5 m) of the cell outline contains about 100 pixels, allowing

measurement ofrto 2.2% accuracy with 95% confidence. The acquisition time for 100

pixels with two polarization states is 2 ms, indicating that 2PPM is capable of

distinguishing between two states differing in rby ~5% in 2 ms. In molecular systems

displaying a larger change in r, the temporal resolution of 2PPM can be well below 1 ms.

Our experimental results (Figs. 4, 5) are in excellent agreement with this analysis.

8/6/2019 nmeth

24/26

Forrmax determination, apart from the photon noise issues analyzed above, noise

and errors stemming from geometrical approximation of cell shape need to be considered.

Our data (Fig. 5d) indicates that the 95%CI forrmax in a single cell is typically 8-12% of

the mean (including 2-3% caused by photon noise). Averaging of several cells allows

narrowing the CI. While the 95%CIs for intracellular calcium concentrations determined

by FRET are typically ~40 smaller than for concentrations determined by 2PPM, this

needs to be considered in context. The FRET determinations used 6,000 longer

illumination time and ~50,000 larger cell area from which quantitative data was being

acquired. Improvements in polarization modulation and image processing software

(allowing using larger parts of cells to be used forrmax determination), and other

enhancements should allow very fast and accurate determinations ofrmax and other

biophysical properties in individual cells.

8/6/2019 nmeth

25/26

SUPPLEMENTARY REFERENCES

1. Hughes, T.E., Zhang, H., Logothetis, D.E. & Berlot, C.H. Visualization of a

functional Galpha q-green fluorescent protein fusion in living cells. Association

with the plasma membrane is disrupted by mutational activation and byelimination of palmitoylation sites, but not be activation mediated by receptors or

AlF4.J Biol Chem 276, 4227-4235 (2001).

2. Hein, P. et al. Gs activation is time-limiting in initiating receptor-mediatedsignaling.J Biol Chem 281, 33345-33351 (2006).

3. Tateyama, M., Abe, H., Nakata, H., Saito, O. & Kubo, Y. Ligand-induced

rearrangement of the dimeric metabotropic glutamate receptor 1alpha.Nat StructMol Biol 11, 637-642 (2004).

4. Krasel, C., Bunemann, M., Lorenz, K. & Lohse, M.J. Beta-arrestin binding to the

beta2-adrenergic receptor requires both receptor phosphorylation and receptoractivation.J Biol Chem 280, 9528-9535 (2005).

5. Lundby, A., Mutoh, H., Dimitrov, D., Akemann, W. & Knopfel, T. Engineeringof a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP

voltage-sensing movements. PLoS One 3, e2514 (2008).6. Feinstein, P., Bozza, T., Rodriguez, I., Vassalli, A. & Mombaerts, P. Axon

guidance of mouse olfactory sensory neurons by odorant receptors and the beta2

adrenergic receptor. Cell 117, 833-846 (2004).7. Roorda, R.D., Hohl, T.M., Toledo-Crow, R. & Miesenbock, G. Video-rate

nonlinear microscopy of neuronal membrane dynamics with genetically encoded

probes.J Neurophysiol 92, 609-621 (2004).8. Bertl, A. et al. Characterization of potassium transport in wild-type and isogenic

yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations.MolMicrobiol 47, 767-780 (2003).

9. Oda, H. & Tsukita, S. Real-time imaging of cell-cell adherens junctions reveals

that Drosophila mesoderm invagination begins with two phases of apicalconstriction of cells.J Cell Sci 114, 493-501 (2001).

10. Leaney, J.L., Benians, A., Graves, F.M. & Tinker, A. A novel strategy to engineer

functional fluorescent inhibitory G-protein alpha subunits.J Biol Chem 277,

28803-28809 (2002).11. Bunemann, M., Frank, M. & Lohse, M.J. Gi protein activation in intact cells

involves subunit rearrangement rather than dissociation. Proc Natl Acad Sci U S A

100, 16077-16082 (2003).12. Hein, P., Frank, M., Hoffmann, C., Lohse, M.J. & Bunemann, M. Dynamics of

receptor/G protein coupling in living cells.Embo J24, 4106-4114 (2005).

13. Gibson, S.K. & Gilman, A.G. Gialpha and Gbeta subunits both define selectivityof G protein activation by alpha2-adrenergic receptors. Proc Natl Acad Sci U S A

103, 212-217 (2006).

14. Frank, M., Thumer, L., Lohse, M.J. & Bunemann, M. G Protein activation

without subunit dissociation depends on a G{alpha}(i)-specific region.J BiolChem 280, 24584-24590 (2005).

8/6/2019 nmeth

26/26

15. Azpiazu, I., Akgoz, M., Kalyanaraman, V. & Gautam, N. G protein betagamma11

complex translocation is induced by Gi, Gq and Gs coupling receptors and isregulated by the alpha subunit type. Cell Signal 18, 1190-1200 (2006).

16. Yu, J.Z. & Rasenick, M.M. Real-time visualization of a fluorescent G(alpha)(s):

dissociation of the activated G protein from plasma membrane.Mol Pharmacol

61, 352-359 (2002).17. Hynes, T.R., Mervine, S.M., Yost, E.A., Sabo, J.L. & Berlot, C.H. Live cell

imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas

and beta1gamma7 internalize upon stimulation and exhibit similar traffickingpatterns that differ from that of the beta2-adrenergic receptor.J Biol Chem 279,

44101-44112 (2004).

18. Zhou, J.Y., Toth, P.T. & Miller, R.J. Direct interactions between theheterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like

domain-containing regulator of G protein signaling 11: gain of function of cyan

fluorescent protein-tagged G gamma 3.J Pharmacol Exp Ther305, 460-466(2003).

19. Rochais, F. et al. Real-time optical recording of beta1-adrenergic receptoractivation reveals supersensitivity of the Arg389 variant to carvedilol.J Clin

Invest117, 229-235 (2007).20. Airan, R.D., Thompson, K.R., Fenno, L.E., Bernstein, H. & Deisseroth, K.

Temporally precise in vivo control of intracellular signalling.Nature 458, 1025-

1029 (2009).21. Hires, S.A., Zhu, Y. & Tsien, R.Y. Optical measurement of synaptic glutamate

spillover and reuptake by linker optimized glutamate-sensitive fluorescent

reporters. Proc Natl Acad Sci U S A 105, 4411-4416 (2008).22. Palmer, A.E. et al. Ca2+ indicators based on computationally redesigned

calmodulin-peptide pairs. Chem Biol 13, 521-530 (2006).23. Han, X. & Boyden, E.S. Multiple-color optical activation, silencing, and

desynchronization of neural activity, with single-spike temporal resolution. PLoS

One 2, e299 (2007).24. Grynkiewicz, G., Poenie, M. & Tsien, R.Y. A new generation of Ca2+ indicators

with greatly improved fluorescence properties.J Biol Chem 260, 3440-3450

(1985).

nmeth

Documents