Imaging nanometer domains of b-adrenergic receptorcomplexes on the surface of cardiac myocytesAnatoli Ianoul1,2, Donna D Grant1, Yanouchka Rouleau1, Mahmud Bani-Yaghoub3, Linda J Johnston1 &John Paul Pezacki1

The contraction of cardiac myocytes is initiated by ligandbinding to adrenergic receptors1,2 contained in nanoscalemultiprotein complexes called signalosomes3. The compositionand number of functional signalosomes within cardiacmyocytes defines the molecular basis of the response toadrenergic stimuli3–6. For the first time, we demonstratedthe ability of near-field scanning optical microscopy tovisualize b-adrenergic receptors at the nanoscale in situ.On H9C2 cells, mouse neonatal and mouse embryonic cardiacmyocytes, we showed that functional receptors are organizedinto multiprotein domains of ~140 nm average diameter.Colocalization experiments in primary cells at the nanometerscale showed that 15–20% of receptors were preassociatedin caveolae. These nanoscale complexes were sufficient toeffect changes in ligand-induced contraction rate without therequirement for substantial changes in receptor distributionin the cellular membrane. Using fluorescence intensitiesassociated with these nanodomains, we estimated the receptordensity within the observed nanometer features and establisheda lower limit for the number of receptors in the signalosome.

A primary signaling pathway that controls the beating rate in themammalian heart is initiated by the binding of catecholamines toG protein–coupled b-adrenergic receptors (bARs) in cardiac myo-cytes1,2. Critical to this signaling cascade is the association of bARsinto multiprotein complexes called signalosomes3 that include the a, band g G protein subunits and either the b1 or b2 isoform of bAR.These signalosomes also appear to have a defined spatial relationshipwith respect to the voltage-gated L-type Ca2+ channels7, which areresponsible for generating the calcium gradient required for myocytecontraction1,2. It has been postulated that upon agonist binding,higher-order signalosome aggregates form, giving rise to signalingplatforms with altered functional properties8,9. Changes in the com-position of signalosomes also modulate bAR function by causingreceptor desensitization10, sequestration of the receptor to subcellularmembranous compartments and internalization3. Although it is clearthat changes in signalosome-aggregation numbers and compositionhave a critical effect on both the cellular physiology and beat rate of

the heart, visualization of these events at the nanoscale has beenchallenging. However, recent advances in scanning probe microscopieshave improved the prospects for high-resolution imaging of mem-brane proteins, permitting the observation of nanoscale complexes, asshown by atomic force microscopy (AFM) of the G protein–coupledreceptor (GPCR) rhodopsin in native disk membrane patches11. Arelated technique, near-field scanning optical microscopy (NSOM),has the ability to detect an optical signal with a lateral spatialresolution beyond the classical diffraction limit12, a feature that allowsfor the imaging of membrane proteins on cell membranes withfluorescence-labeling techniques. The nanometer-scale topologicaland optical information that can be obtained with NSOM has thepotential to provide unique insight into the distribution of integralmembrane proteins on cell surfaces, including those involved incontrol of heart beat rate13,14. NSOM can also determine the number,density and distribution of receptors on the surface of cardiacmyocytes and elucidate changes during the course of signaling events,allowing us to better understand the molecular mechanisms of signaltransduction. Such information can aid in the development of small-molecule therapeutics to modulate signal transduction. Here, we havedemonstrated the ability of NSOM to image bAR complexes, estimatethe stoichiometry of bAR in the signalosome and identify themolecular components associated with active complexes.

We first used NSOM to investigate the expression of b2AR on H9C2cells, a clonal cardiac cell line derived from embryonic rat hearts, asthis line shows cardiac muscle characteristics and has been used tostudy cardiac ion channel function15. Using bent-fiber NSOM probeswith an aperture diameter of 40–80 nm operating in the tapping modeand fluorescence microscopy, we examined the expression and dis-tribution of b2AR immunostained with primary anti-b2AR andfluorescently labeled secondary antibody on the plasma membraneof H9C2 cells. Cells were seeded on glass cover slips and fixed, andNSOM images were acquired as previously described14. Representativeimages collected by fluorescence microscopy and NSOM of b2AR onH9C2 cells are shown in Figure 1. The superior resolution of theNSOM images relative to the conventional fluorescence images allowsvisualization of discrete b2AR clusters (Fig. 1b) that are uniformlydistributed on the plasma membrane. In addition to the increased

Published online 7 August 2005; doi:10.1038/nchembio726

1The Steacie Institute for Molecular Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, Canada K1A 0R6. 2Present address:Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Canada, K1S 5B6. 3The Institute for Biological Sciences, National ResearchCouncil Canada, 1200 Montreal Road, Bldg M-54, Ottawa, Canada K1A 0R6. Correspondence should be addressed to J.P.P. (john.pezacki@nrc-cnrc.gc.ca) orL.J.J. (linda.johnston@nrc-cnrc.gc.ca).

19 6 VOLUME 1 NUMBER 4 SEPTEMBER 2005 NATURE CHEMICAL BIOLOGY

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spatial resolution, NSOM has the advantageof monitoring only membrane-associatedproteins. NSOM is very specific in its abilityto visualize fluorescently tagged molecules atthe cellular membrane; fluorescent molecules100 nm below the membrane surface will beinvisible14. The levels of nonspecific bindingof the secondary antibody and contributionsfrom topography-induced artifacts for theNSOM images were determined by imagingof cells that were fixed and stained only withthe secondary antibody. Comparisons oftopography and fluorescence intensitiesfor several images showed results similar toprevious studies with H9C2 cells; signals dueto the sum of topography-induced artifactsand nonspecific binding were determinedto be o10% of the average fluorescence-intensity signal14.

We analyzed the cluster size and densityfor several cells from several different pre-parations using methods described pre-viously14. Analyses of seven individualimages (Z20 � 20 per mm2) gave an averagecluster density of 1.0 features per mm2. Theseb2AR clusters were mostly circular in shapeand varied in diameter from several hundred nm to 40–80 nm.Detailed analyses of several small-scale images of b2AR-stainedH9C2 cells yielded a distribution for b2AR clusters (Fig. 1g) with anaverage cluster diameter of approximately 140 nm. Although the sizeof the smallest feature was determined by the diameter of the probeaperture (B60 nm), these smallest-scale features represented less than10% of the imaged protein complexes.

The number of receptor molecules per cluster provides importantinsight into the molecular nature of the signalosomes that lead tochanges in cardiac myocyte contraction rates. We estimated thenumber of receptors within observed nanometer-scale clusters fromthe total fluorescence intensity using the average intensity for a single

secondary antibody as a calibration. NSOM images of antibodies atvarious dilutions on mica were used to assess the average fluorescenceintensity for the secondary antibody (4–6 dye molecules per antibody,Supplementary Fig. 1 online). However, the number of observedantibodies per cluster represents a lower limit for the number ofreceptors present. Relating the number of secondary antibodies percluster to the receptor number requires assumptions concerning thestoichiometry of binding for both primary and secondary antibodies.If receptors are organized into dimers16, the binding stoichiometry forprimary antibody will be between 0.5:1 and 1:1, depending on whethera single antibody can bind simultaneously to two receptors. Cross-linking of primary antibodies by the secondary or incomplete labeling

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Figure 1 Confocal fluorescence and NSOM

images for fixed H9C2 cells immunostained

for b2AR, b1AR or caveolin-3. The cell nucleus

is counterstained with DAPI (shown in blue)

for all the fluorescence images. (a) b2AR

fluorescence. (b) 40 � 40 mm2 NSOM image

(left) showing an overlay of topography and

b2AR fluorescence and a small-scale NSOM

fluorescence image (right) of the boxed region.

The highlighted features in the small-scale

image correspond to 2–4 receptors (circle),

15–30 receptors (square) and 36–72

receptors (triangle). (c) b1AR fluorescence.

(d) 20 � 20 mm2 NSOM image (left) showing an

overlay of topography and b1AR fluorescence anda small-scale NSOM fluorescence image (right).

(e) Calcium efflux measured according to

standard procedures7,28. (f) NSOM images

for b2AR for cells treated with isoproterenol.

(g) histograms representing average cluster size

distributions of b1AR (right) and b2AR (left) on

H9C2 cells. (h) NSOM topography (left) and

fluorescence images for b2AR (middle) and

caveolin-3 (right).

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will further decrease the secondary antibody–to-receptor ratio.Furthermore, washing steps during incubation of fixed cells withprimary and secondary antibodies certainly lower the number ofbound antibodies. To address the issue of quantitative labeling ofthe receptor, we also derived a stable cell line expressing a fusionprotein of b2AR and green fluorescent protein (GFP) and tested ourantibody’s ability to label receptor in this cell line. Images that weobtained from this cell line show that there is reasonable colocalizationof the fusion protein and the primary-secondary antibody complex,suggesting a high degree of antibody labeling under the conditionsused for this study (Supplementary Figs. 2 and 3 online). Consideringthe above factors, we believe that a conservative approximation for thesecondary antibody–to-receptor ratio is between 0.5:1 and 1:1. Weused this range in combination with the fluorescence intensities toestimate the number of receptors for individual clusters. The smallestfeatures detected correspond to one to four receptors. We observedclusters with a range of intensities; those varying in diameter from 120to 160 nm corresponded to anywhere from 12 to 72 molecules ofreceptor (Fig. 1b and Supplementary Fig. 1).

To understand the importance of receptor packing density onadrenergic signal transduction, we compared the packing densitiesof b2AR with that predicted for the photoreceptor rhodopsin. We usedthe cluster densities and intensities for average-sized clusters toestimate that there are approximately 30–50 b2AR receptors permm2 in H9C2 cells. This compares well with previous estimates ofb2AR density for cardiac myocytes17 and A549 cells18 (30 and 19.9receptors per mm2, respectively) indicating that the assumptionsinvolved in quantifying the receptor numbers are reasonable. Ourpredicted receptor densities are considerably lower than the maximumpacking density of 6.29 � 104 proteins per mm2 estimated by AFM forrhodopsin in native membrane patches11. In fact, if b2AR receptorsrequired the packing density observed by AFM imaging of rhodopsinon native membrane patches, then we predict that there would beapproximately 1,000 receptor molecules per average 140-nm cluster.Even the most conservative estimates for the labeling efficiencies ofantibodies used to visualize b2AR receptors would still place thenumber of b2AR receptors per average 140-nm cluster substantiallylower than this theoretical maximum. It is likely that the requirementfor efficient light absorption by rhodopsin may lead to a high packingdensity relative to other GPCRs. Lower packing densities observed forb2AR may reflect the relative efficiency in signal transduction andpossibly spatial requirements for functional signalosome assembly forb2AR. Previous observations suggest that b2AR receptor homodimer-ization occurs early during the trafficking of the receptors to theplasma membrane and that the dimer may be important to subse-quent adrenergic signaling16,19. From the receptor densities observed,it is clear that there is more than enough room for individual receptormolecules to freely diffuse throughout the area of each nanoscalecluster without the requirement for oligomerization. On the otherhand, the preorganization of multiple bAR molecules into stablenanometer clusters may provide a mechanism for the rapid responseof cardiac myocytes to b-adrenergic agonists by circumventing therequirement for large-scale diffusion of receptor molecules in theadrenergic response.

Because the b1 isoform of the adrenergic receptor is also coupled tothe Gs/cAMP pathway and can induce ionotropic effects in murineneonatal myocytes, we performed the same series of experiments usingprimary anti-b1AR antibodies to localize the b1AR. We collected bothfluorescence and NSOM images of b1AR on the plasma membrane ofH9C2 cells (Fig. 1c,d). Both imaging techniques revealed a lessuniform distribution of b1AR versus that of b2AR on the plasma

membrane of H9C2 cells. A substantial amount of b1AR appearedanchored within the plasma membrane over the nuclei of the H9C2cells (Fig. 1c,d). This observation is consistent with previous reports ofdifferences in spatial distribution and resulting differences in functionfor b1AR and b2AR (refs. 5,20). We analyzed several independenthigh-resolution NSOM images and determined that the average size ofb1AR clusters on H9C2 cells was approximately B100 nm (Fig. 1),which is slightly smaller than the average b2AR cluster size. Wedetermined that the average density was higher than that for b2ARat B3.1 features per mm2. Assuming similar packing densities percluster and taking into account the small reduction in cluster size forthe b1AR, the density of b1AR would still be higher than that of b2AR.The higher density of b1AR compared with b2AR is consistent withbiochemical investigations that predict a relative ratio of the twoisoforms to be B3:1 in favor of b1AR (ref. 20). However, in contrast tothe largely uniform receptor-complex density observed for b2AR, thedensity of complexes containing b1AR varied by over a factor of 2depending on the image size and area of the cell imaged (Supple-mentary Table 1). Nonuniform distributions of membrane proteinssuch as b1AR require the collection of a larger number of representa-tive small-scale images to fully characterize the cluster size and density.

There is an increasing amount of evidence suggesting that receptoroligomerization has an important role in signal transduction and maymodulate the size and composition of signalosome protein com-plexes7. Although bioluminescence resonance energy transfer experi-ments suggest that these receptor interactions may be needed forsignaling19, other studies have raised the possibility that receptoroligomerization is the consequence of localization to a specificsubcellular compartment16,21. Because the role of receptor oligomer-ization in the adrenergic signaling pathway is not well understood, wesought to investigate the possibility that changes in the number anddistribution of receptor molecules could occur in stimulated H9C2cells on the nanoscale. The presence of the expected adrenergicresponse in this cell line was confirmed by the two-fold increase incalcium efflux (Fig. 1e) upon isoproterenol treatment. We collectedNSOM images of b1AR or b2AR under a variety of conditionson cardiac myocytes fixed and stained between 0 and 10 minutesafter adrenergic stimulation (Fig. 1f). Analysis of cluster size andreceptor density revealed no detectable difference for either b1 or b2

after isoproterenol treatment. Therefore, large-scale changes in signa-losome cluster size and localization are not required to affect theadrenergic response.

Because phenotype, localization and bAR signalosome compositioncan be altered in immortalized cell lines as compared with primary celltypes21–23, we next investigated the expression of adrenergic receptorsand the effect of adrenergic stimulation on primary murine cardiacmyocytes (Fig. 2) to determine whether our results with H9C2 cellsreflected the true physiological state of b2AR localization. We focusedon the b2AR isoform because it is hypothesized to be more importantfor growth and development than other isoforms in the developingmurine heart21,23. Both embryonic and neonatal cells were isolateddirectly from CD-1 mice (embryonic day 12–14 and postnatal day5–9) and cultured on NSOM glass coverslips. These cells grew infunctional beating clusters, and the adrenergic response was confirmedby beating-rate assays in the presence and absence of isoproterenolligand (Fig. 2a).

We confirmed the expression of b2AR by fluorescence microscopyof immunostained neonatal and embryonic (Fig. 2a,c) mouse cardiacmyocytes in all cases. NSOM imaging of beating neonatal andembryonic (Fig. 2b,d) myocytes that were fixed and immunostainedrevealed a pattern and size of clustering similar to those obtained on

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H9C2 cells. We determined that the average cluster size of b2ARs onneonatal and embryonic cells was B150 nm. The average density ofthese features was determined to be 0.67 features per mm2, which wasslightly lower than the average density observed in the immortalizedcell line. Only minor differences in cluster size and number wereobserved between murine myocyte cells that were embryonic day12–14 and neonatal day 5–9, suggesting that b2AR’s role in growth anddevelopment is probably not linked with its expression levels or alteredmembrane localization during embryonic development24.

We also conducted NSOM and fluorescence-microscopy experi-ments of b2ARs in mouse neonatal and embryonic cardiac myocytesduring the initial phase of adrenergic response to the catecholamineisoproterenol. Cells were fixed and imaged before detectable ligand-mediated receptor endocytosis occurred16. Image analysis showed thatcluster size and number were not altered substantially upon adrenergicstimulation (Fig. 2b), in agreement with results in H9C2 cells.

Furthermore, we did not observe largechanges in fluorescence signal intensities percluster following adrenergic stimulation inany of the cell types studied. Images of cardiacmyocytes before and after adrenergic stimula-tion that were collected with the same NSOMprobe and excitation intensity did not showdifferences in fluorescence intensity for indi-vidual clusters that were larger than the cell-cell and image-image variation typicallyobserved. Together these observations suggestthat, in primary as well as the H9C2 cells,b2AR receptors are located in signalosomeclusters that are already preassociated beforethe hormone-initiated signal cascade. TheNSOM images of b2AR on mouse neonataland embryonic cells represent the first directobservations of nanoscale-sized clusters ofbARs on the surfaces of primary cardiacmyocytes and highlight the potential of thisimaging technique to be adapted to a widevariety of technically challenging systems.

Several key components of b2AR signalo-somes can be found in membrane fractionsthat are rich in caveolin proteins (b2AR, Gi2a,Gi3a, adenylate cyclase subunits 5/6, compo-nents of PKA and domains of elevatedcAMP)5,25. Co-immunoprecipitation ofcaveolin-3 with b2AR, filipin sensitivity for

b2AR signaling, and studies of b1AR and b2AR knockout mouseneonatal cells strongly suggest that caveolar localization of b2AR isessential for physiological signaling21. To determine the localizationpattern of caveolae, we immunostained H9C2 cells using a mono-clonal antibody to caveolin-3, then stained with a fluorescent second-ary antibody. NSOM imaging of these cells (Fig. 1h) indicated that thecaveolin-3 cluster density on the cellular membrane is approximatelytwice that of b2AR, similar to results that were obtained with NSOMimages of the a1c-subunit of the voltage-gated L-type Ca2+ channel14.We performed similar experiments with neonatal mouse cardiacmyocytes (Fig. 2e). We performed extensive analyses of the imagesobtained with the primary cells and determined that the averagecluster size was similar to that of the b2AR complexes at B145 nm,whereas the average density of these clusters was substantially higherat 4.1 features per mm2. Given that the size of these complexes issimilar to the size of caveolae as visualized by electron microscopy

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Figure 2 Confocal fluorescence and NSOM

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myocytes immunostained for b2AR and caveolin-

3. Each set of NSOM images shows topography

and fluorescence images recorded simultaneously

for the same area of the sample. (a) b2AR

fluorescence and contraction data for neonatal

myocytes measured according to established

procedures29,30. (b) NSOM topography and b2AR

fluorescence for neonatal myocytes untreated

(upper) and followed by isoproterenol treatment

(lower). (c) Fluorescence for b2AR in embryonic

myocytes. (d) NSOM topography (left) and b2AR

fluorescence (right) for embryonic myocytes.

(e) NSOM topography (left) and caveolin-3fluorescence (right) for embryonic myocytes.

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(50–100 nm; ref. 3) and that caveolin-3 is a major constituent ofcaveolae, each cluster most likely represents a single caveola. Threefactors that might contribute to a size overestimation of caveolae inNSOM experiments are tip convolution, antibody size and the altereddepth of proteins localized within the invaginated membrane ofcaveolae. The imaging experiments will be most sensitive to changesin the depth of the caveolin-3 signal resulting from the morphologyof caveolae14.

The high-resolution capabilities of NSOM should make it possiblefor colocalization experiments to be conducted on the nanoscale.NSOM has several advantages, including the ability to measure clustersizes down to 50 nm, the ability to spatially resolve clusters that aretoo close together to be separated at the maximum half-wavelengthspatial resolution of confocal microscopy and the ability to selectivelyimage receptors at the membrane rather than those that are inter-nalized. To determine whether b2ARs are localizing to the samemultiprotein clusters as caveolin-3, we performed fluorescence micro-scopy and NSOM on neonatal cardiac myocytes immunostained withrabbit anti-b2AR and goat anti-caveolin-3 antibodies and Cy3 anti-rabbit and Cy2 anti-goat secondary antibodies (Fig. 3). Our analysesof NSOM images of the doubly stained neonatal cells revealed thatboth b2AR and caveolin-3 localize to clusters with average sizes anddistribution densities identical to those previously observed withsingly stained cells. In addition, signal overlap for the Cy3 andCy2 secondary antibodies was observed within a subpopulation ofclusters, showing that caveolin-3 and b2AR colocalize to the samemultiprotein domain. Detailed analysis of colocalization data from

two independent NSOM images recordedwith a B40-nm tip based on Manders coeffi-cients26 indicated that approximately 15–20%of b2AR is associated with caveolin-3 (Fig. 3b,Supplementary Figs. 4 and 5 online). Weinterpret these features as representing b2ARcolocalizing with single caveolae on theplasma membrane. Interestingly, a substantialfraction of b2AR that are not colocalized withcaveolin-3 appear to be located near caveolae,possibly reflecting membrane protein traffick-ing into and out of caveolae.

The localization of bARs to caveolae isconsistent with previous results from lowbuoyant density sucrose gradient experimentsand colocalization studies based on confocalfluorescence microscopy4–6. Because it hasbeen shown that bAR stimulation requiresthe presence of caveolae, these results suggestthat even in the absence of adrenergicstimulation, functionally competent bARcomplexes are preassembled in or near caveo-lae. Our data support the hypothesis thatlocalization of bARs to caveolae provides afunctional platform that can promote efficientinitiation of signal transduction3–6. Ourobservations are also consistent with recentevidence indicating that, for the b2AR iso-form, the caveolar association is inhibitoryto signal transduction and the adrenergicresponse requires trafficking of the b2ARsignalosome out of caveolae before signaltransduction4–6,21. In addition, the lackof complete colocalization of bAR with

caveolin-3 raises the possibility that diverse functional properties ofthe b2AR could arise from its association with multiprotein complexesof different compositions that may not be caveolar in nature. Furthercolocalization studies based on NSOM are currently under way toestablish the nature of these noncaveolar complexes and whether anychanges in the distribution of caveolar-associated bARs are promotedby adrenergic agonists. We established the utility of NSOM-basedimaging techniques to resolve nanometer-scale complexes on theplasma membrane of both primary and immortalized cell lines,showed colocalization within these complexes and estimated thestoichiometry of the labeled molecular components in situ. NSOMshould in principle be widely applicable to imaging and functionalcharacterization of a diverse range of signalosomes and the pharma-cological effects of agonists and antagonists on their subcellularlocalization, internalization and aggregation.

METHODSCell culture. H9C2 cells (ATCC) were grown in Dulbecco’s modified Eagle’s

medium (Gibco/Invitrogen) supplemented with 10% fetal bovine serum (FBS),

glutamine (2 mM), penicillin (100 IU ml�1), and streptomycin (100 mg ml�1)

under standard culture conditions (37 1C, 5% CO2). Primary cells were

maintained with DME and 10% FBS.

Cardiomyocytes isolation. CD-1 pregnant mice (Charles River Laboratories)

were anesthetized (E14–E19) to remove the fetuses. The fetal hearts were

dissected out, the blood was pinched out and the hearts were washed three

times with Earle’s salt solution (cold). They were suspended in 1% trypsin in

PBS, chopped into small pieces and incubated at room temperature for 10 min.

Topography Caveolin-3 (Cy2) β2AR (Cy3)

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Figure 3 Confocal fluorescence and NSOM images for the colocalization of b2AR with caveolae on

neonatal cardiac myocytes. (a) Confocal fluorescence images for fixed neonatal cardiac myocytes

immunostained for b2AR (red) and caveolin-3 (green) with a large-scale overlay image showing

areas of colocalization in yellow. (b) Corresponding NSOM images for a similarly stained myocyte

with individual topography (right) and caveolin-3 (right) and b2AR (middle) fluorescence (top) and

an overlay image (bottom) showing colocalization (yellow) of b2AR (red) with caveolin-3 (green) on a

nanometer scale.

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Five milliliters of DME plus 10% FBS were added and pipetted up and down

12 times. The cell suspension was passed through a 40-micron filter and spun

at 180g for 5 min. The supernatant was removed and the cell pellet was

resuspended in 7 ml DME plus 10% FBS. A viability cell count was then

performed, and the cells were plated at 1 � 106 cells per ml in DME plus 10%

FBS on gelatin-coated dishes and coverslips. The medium was changed after

24 h, and the cells were fed three times per week. To coat the plates or coverslips

with gelatin, a 0.1% gelatin solution in H2O is added to cover the bottom of the

tissue culture dish of the coverslip and incubated 1 h at room temperature.

Then, the gelatin solution is removed and the cells are plated. Animal protocols

used in this study were approved by the Canadian Council on Animal Care.

Antibody staining. The cells were washed once with PBS pH 7.4, fixed with

3.7% formaldehyde for 30 min at 4 1C and washed three times with PBS to

remove the excess formaldehyde. The PBS covering the cells that were fixed on

18-mm round glass coverslips was removed. One well was covered with 1 ml of

PBS, which served as the no-primary-antibody control. The remaining mono-

layers were covered with a 0.5-ml solution of the corresponding primary

antibody dilution. The antibodies used were rat polyclonal anti-b1AR and anti-

b2AR (Santa Cruz), with stock solutions at 0.2 mg ml�1 and both used at a

1:250 dilution in 1� PBS, and mouse monoclonal anti-caveolin-3 (BD

Biosciences), with a stock of 0.25 mg ml�1 used at 1:300 dilution in 1X PBS.

The multiwell plate was sealed with parafilm, covered with foil and stained at

4 1C overnight. The primary antibody solution was aspirated and the cells were

washed three times for 3 min with 1 ml PBS. All monolayers were then covered

with 0.5 ml of secondary antibody solution, Cy2 anti-mouse (0.14 mg ml�1

stock solution) and/or Cy3 anti-rabbit (0.15 mg ml�1 stock solution; both

antibodies from Jackson ImmunoResearch Laboratories, Inc.) used at a 1:200

dilution in 1X PBS and stained at room temperature for 45 min to 2 h. The

secondary antibody solution was then aspirated, and the cells were washed

three times for 3 min with 1 ml PBS. For fluorescence microscopy imaging, the

monolayers were covered with 0.5 ml of 300 nM DAPI solution (Molecular

Probes) and stained for 2 min at room temperature. The monolayers were then

washed three times for 3 min with 0.5 ml 1X PBS. After the final wash, the

monolayers were overlayed with 0.5 ml of 50% glycerol to keep the cells

hydrated. For NSOM imaging, after the three washes with 1X PBS, the cells on

18-mm coverslips were rinsed three times with distilled autoclaved water and

allowed to air-dry for a minimum duration of 12 h before imaging.

Fluorescence imaging of fixed cells. Fluorescence images were collected with a

ZeissAxiovert 200M inverted microscope with Axiovision 3.1 software. The

cells were photographed with an AxioCam connected to the inverted micro-

scope. We performed confocal experiments for all cell types in parallel

with each NSOM experiment in order to confirm the specificity of antibody

labeling, and to be sure that staining density and intensity remained the same

under all circumstances.

Calcium assay. Quantification of intracellular calcium was done with the long-

wavelength calcium indicator Oregon Green BAPTA-1/AM (Molecular Probes).

In brief, the cells were trypsinized and resuspended in Krebs-HEPES buffer

(118 mM NaCl, 4.2 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM

KH2PO4, 11.7 mM D-glucose, 1.3 mM CaCl2, 10 mM HEPES, pH 7.4). The

cells were counted by Trypan Blue Exclusion (Invitrogen), resuspended at

1 � 106 cells per ml in Krebs-HEPES buffer containing 1% Pluronic and

5 mM Oregon Green and incubated for 1 h at 25 1C. The cells were spun down

at 180g for 5 min and resuspended in 1 ml of Krebs-HEPES buffer

containing 0.5% BSA. The cells were counted by Trypan Blue Exclusion. The

test compounds (for example, 10 mM isoproterenol) were added to the wells of

a shielded-wall, clear-bottom plate. A total of 100,000 cells were transferred per

well in 100 ml of Krebs-HEPES buffer containing 0.5% BSA. With an excitation

wavelength of 488 nm, fluorescence was read at 585 nm (F). Ten microliters of

10% Triton-X100 was added to all wells and mixed gently with pipet tips to lyse

the cells. The plate was read again (Fmax). Ten microliters of 110 mM EDTA was

added to chelate calcium and the plate was read again (Fmin).

NSOM. Bent NSOM probes were prepared from high GeO2-doped fibers with

a core diameter of 3 mm via a two-step chemical etching method followed by

aluminum deposition and focused ion-beam milling to produce a flat circular

aperture27. Probes with aperture diameters between 110 and 40 nm were used

in the present work (estimated from SEM images and by imaging 20-nm dye-

labeled polymer microspheres). The estimated spring constant for these probes

is B100 N m�1 (ref. 27). NSOM experiments were carried out on a combined

AFM/NSOM microscope based on a Digital Instruments Bioscope mounted on

an inverted fluorescence microscope (Zeiss Axiovert 100), with cellular imaging

performed as described previously with either 488 (Cy2) or 568 nm (Cy3)

excitation14. The incident laser intensity on the probe and the APD counts were

routinely measured for individual probes to ensure that similar excitation

intensities were used with different NSOM probes. Cluster-size analysis was

performed based on original nonprocessed NSOM images with custom-made

software that determines the number of clusters and their location in the image,

as well as their height (intensity) and half-width. The software allows for

smoothing and background subtraction to remove weak background signals.

All reported cluster sizes are for small-scale images (r10 � 10 mm2) for which

the pixel size is notably smaller than the probe aperture. Reported sizes are a

convolution of feature and probe-aperture size. NSOM colocalization data were

analyzed with Image J software (Supplementary Methods online). NSOM

imaging of cells was carried out as soon after mild fixation and drying as

possible in order to minimize potential artifacts.

ACKNOWLEDGMENTSL.J.J. and J.P.P. thank the Genomics and Health Initiative of the NationalResearch Council Canada for partial financial support for this research. We thankR. Trembley for assistance in primary cell isolation, Z. Lu for NSOM imagingsupport, D. Moffatt for assistance with cluster analysis software, and R. Taylorand N.K. Goto (University of Ottawa) for useful discussions.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 6 April; accepted 18 July 2005

Published online at http://www.nature.com/naturechemicalbiology/

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