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Neuroscience 144 (2007) 135–143

0d

ANOSCALE ORGANIZATION OF NICOTINIC ACETYLCHOLINEECEPTORS REVEALED BY STIMULATED EMISSION

EPLETION MICROSCOPY

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. R. KELLNER,a C. J. BAIER,b K. I. WILLIG,a

. W. HELLa* AND F. J. BARRANTESb

Max Planck Institute for Biophysical Chemistry, Department ofanoBiophotonics, 37077 Göttingen, Germany

UNESCO Chair of Biophysics & Molecular Neurobiology and Institutoe Investigaciones Bioquímicas, 8000 Bahía Blanca, Argentina

bstract—Acetylcholine receptor (AChR) supramolecular ag-regates that have hitherto only been accessible to examina-ion by electron microscopy were imaged with stimulatedmission depletion (STED) fluorescence microscopy, provid-ng resolution beyond limits of diffraction of classical wide-eld or confocal microscopes. We examined a Chinese ham-ter ovary cell liner CHO-K1/A5, that stably expresses adulturine AChR. Whereas confocal microscopy displays AChR

lusters as diffraction-limited dots of �200 nm diameter,TED microscopy yields nanoclusters with a peak size dis-

ribution of �55 nm. Utilizing this resolution, we show thatholesterol depletion by acute (30 min, 37 °C) exposure toethyl-�-cyclodextrin alters the short and long range orga-ization of AChR nanoclusters on the cell surface. In thehort range, AChRs form larger nanoclusters, possibly re-

ated to the alteration of cholesterol-dependent protein–pro-ein associations. Ripley’s K-test on STED images revealshanges in nanocluster distribution on larger scales (0.5–.5 �m), which possibly are related to the abolition of cy-oskeletal physical barriers preventing the lateral diffusion ofChR nanoclusters. © 2006 IBRO. Published by Elsevier Ltd.ll rights reserved.

ey words: cyloskeleton, supramolecular aggregates, cho-esterol, synapse, ion channel, STED microscopy.

ast synaptic transmission as mediated by the neurotrans-itter acetylcholine imposes concentration and spatial re-uirements to achieve high efficacy. The high density oficotinic acetylcholine receptors (AChRs), tightly aggre-ated in the form of micron-sized two-dimensional clusters,

s a characteristic feature of the postsynaptic membrane ofnnervated adult neuromuscular junctions (NMJ) (Sanesnd Lichtman, 2001). Postsynaptic maturation can occur,nd clusters form, in the absence of nerve (Kummer et al.,004), but it is not known how these supramolecular ag-regates are constructed at the cell surface in the absence

Corresponding author. Tel: �49-551-2012501; fax: �49-551-2012505.-mail address: hell@nanoscopy.de (S. W. Hell).bbreviations: AChR, nicotinic acetylcholine receptor; �BTX, � bunga-

otoxin; CDx, methyl-�-cyclodextrin; CI, confidence interval; CSR, com-lete spatial randomness; fix–label, fixation followed by labeling; FWHM,ull-width-half-maximum; label–fix, labeling followed by fixation; NMJ,euromuscular junction; STED, stimulated emission depletion; TMA-

aPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-tolu-nesulfonate.

306-4522/07$30.00�0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2006.08.071

135

f innervation (Willmann and Fuhrer, 2002; Sanes andichtman, 2001). Before establishment of these postsyn-ptic specializations, AChRs shift from a diffusely dis-ersed to a submicron-sized cluster distribution during thearly stages of embryonic development of the NMJ (Will-ann and Fuhrer, 2002; Sanes and Lichtman, 2001).hese marked changes in AChR supramolecular organi-ation occur within a very narrow time window in ontogeny,etween embryonic stages E13 and E14 (Sanes and Li-htman, 2001).

Due to their diffraction-limited resolving power, epiflu-rescence and confocal microscopes fall short of resolvinghe fine structure of these small clusters. However, recentdvances in physical optics have shown that the diffraction

imit of far-field fluorescence microscopy can be overcomey applying the principles of stimulated emission depletionSTED) (Hell and Wichmann, 1994; Klar et al., 2000; re-iewed in Hell, 2003). STED is a member of a new familyf microscopy concepts that, despite using regular lenses,ntails diffraction-unlimited resolution (Hell, 1997, 2004).

n STED microscopy the excitation beam is overlappedith a second, red-shifted doughnut-shaped beam thate-excites fluorophores by stimulated emission. Spatialnd temporal alignment of the excitation focal spot with thee-exciting beam ensures that fluorescence originatesnly from the central area of the excitation spot where theower of the doughnut beam is close to zero (Supplemen-ary Fig. S1). By increasing the intensity of the de-excitingeam beyond a saturation threshold, the resulting fluores-ent spot of the STED microscope can be narrowed downo the molecular scale (Hell, 1997, 2003).

Here we apply STED microscopy to visualize the or-anization of AChR supramolecular aggregates below theiffraction resolution limit. To this end cell-surface AChRsere studied in CHO-K1/A5 cells, a heterologous mamma-

ian cell expression system obtained in one of our labora-ories. CHO-K1/A5 is a clonal cell line stably expressingubstantial amounts of adult murine AChR devoid of non-eceptor anchoring proteins (Roccamo et al., 1999), andhus constituting an excellent model system to study theistribution and organization of this cell-surface receptor.

EXPERIMENTAL PROCEDURES

ell culture

HO-K1/A5 cells were grown in Ham’s F12 medium supple-ented with 10% fetal bovine serum (FBS) for 2–3 days at 37 °C

s in Roccamo et al. (1999) before cytochemical experiments.

ved.

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R. R. Kellner et al. / Neuroscience 144 (2007) 135–143136

reparation of single plasma membrane sheets

or preparation of membrane sheets, CHO-K1/A5 cells wererown on polylysine-coated glass coverslips and disrupted asescribed by Avery et al. (2000) using a 300 ms ultrasound-pulse

n ice-cold KGlu buffer (20 mM Hepes, pH 7.2, containing 120 mMotassium glutamate and 20 mM potassium acetate).

holesterol depletion of cells and single plasmaembrane sheets

HO-K1/A5 cells and plasma membrane sheets, on the sameoverslips, were treated with 10 mM methyl-�-cyclodextrin (CDx;igma Chemical Co., St. Louis, MO, USA) in KGlu-buffer for 20in at 37 °C to deplete their cholesterol content.

luorescence labeling

n order to stain only cell-surface AChRs, CHO-K1/A5 cells wereabeled with fluorescent Alexa594-�-bungarotoxin (�BTX) (Molec-lar Probes, Eugene, OR, USA) or with the monoclonal anti-AChRntibody mAb210 (Sigma Chemical Co.), followed by staining withecondary antibodies previously conjugated to the dye Atto 532.he latter fluorochrome (provided by K. H. Drexhage, Chemistryepartment, University of Siegen, Siegen, Germany) was coupled

o an affinity purified sheep anti-mouse IgG (Jackson ImmunoRe-earch Laboratories, West Grove, PA, USA) via its succinimidylster.

In the �BTX staining procedure, cells and membrane sheetsere stained with Alexa594-�BTX for 1 h at 4 °C in KGlu bufferontaining 1% BSA. In the antibody staining procedure, cells andembrane sheets were incubated with mAb210 for 1 h at 4 °C,

epeatedly washed, and labeled with sheep anti-mouse conju-ated with the green-emitting dye Atto 532 for 1 h at 4 °C. Whensing antibodies, three different conditions were employed: In thefix-label” condition, samples were fixed with 4% paraformalde-yde before labeling, in order to avoid AChR crosslinking (Tzartost al., 1987). Secondly, in the “label-fix” condition, fixation followed

abeling to allow for antibody-induced AChR patching. Thirdly,hen the effect of cholesterol depletion was studied (“CDx-label-x”), samples were treated with CDx and stained with antibodiess described above and fixed. Finally samples were mounted inowiol (6 g Glycerol AR (#4094, Merck, Darmstadt, Germany),.4 g Mowiol 4–88 (Hoechst, Frankfurt, Germany), 6 ml water,2 ml 0.2 M Tris buffer, pH 7.2) and imaged.

ide-field fluorescence microscopy

mages of whole cells and single membrane sheets obtained byltrasound treatment (see below) were obtained using conven-ional epifluorescence microscopy. In some experiments imagingas first undertaken in PBS containing 25 �l/ml of a saturated-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-tolu-nesulfonate (TMA-DPH) solution (Molecular Probes) in PBS. TMA-PH allows one to visualize the lipid moiety of membranes and thus

acilitate the identification of the ultrathin membrane sheets using aikon E-300 fluorescence microscope with a 100�1.4 N.A. oil im-ersion objective driven by a piezoelectric nanopositioning system

PI Instruments GmbH, Karlsruhe, Germany). Imaging was accom-lished with Hamamatsu Orca camera driven by MetaMorph 5.0oftware. Appropriate dichroic and emission filters were employed tovoid crossover of fluorescence emission.

TED microscopy

yes were excited with a laser diode (Picoquant, Berlin, Ger-any) emitting 100 ps pulses at 490 nm wavelength. The diodeas triggered by the STED pulses delivered by an optical

arametric oscillator (OPO, APE, Berlin, Germany), which was o

ynchronously pumped by a Ti:Sapphire laser (MaiTai, Spectrahysics, Mountain View, CA, USA) operating at 80 MHz. TheTED pulses with a wavelength of 615 nm were stretched to00 ps by dispersion in a glass fiber and converted into a focaloughnut by means of a spatial light modulator (Hamamatsu,amamatsu City, Japan) delivering a helical phase ramp (0 –�). Using custom-designed dichroic mirrors both beams wereoupled into an oil immersion lens (HCX PL APO, 100�, Leicaicrosystems, Mannheim, Germany). The typical averageower of the excitation and the STED beam at the sample was�W and 14 mW, respectively. The fluorescence collected by

he lens was imaged onto a counting avalanche photodiode,ith an opening diameter of 71% of that of the back-projecteduorescence spot. STED-imaging was obtained by piezo-scan-ing the sample at a pixel dwell time of �0.3 ms with a pixelpacing of 15 nm�15 nm. The effective point spread function ofhis system was determined experimentally by imaging glass-dsorbed, point-like primary antibodies stained with Atto532

abeled secondary antibodies. Lorentzian fits on the intensityrofiles revealed a full-width-half-maximum (FWHM) of0.8�2.3 nm (mean�S.E.M., data not shown), which is approx-

mately the lateral resolution of this setup.

ata analysis

rightness and FWHM of individual AChR nanoclusters werevaluated with macros written in MatLab (The Mathworks Inc.,atick, MA, USA) in 7.5�7.5 �m2 images having a pixel spac-

ng of 15 nm�15 nm. The user interactively defines a regionnclosing the dot of fluorescence in the image (see Fig. S2 inupplementary material). The background level and the FWHMf the focal spots in the x- and y-direction were derived fromorentzian fits. The brightness was defined as the backgroundorrected sum over all pixels within the FWHM. Averages of thealues for the dots in the x- and y-directions were retained forach dot and exported for statistical analysis. To ensure thatWHM measurements were not affected by dots that werelose together, a stringent chi2 cutoff of less than a 0.02 differ-nce between the fit and the data was placed on the Lorentziant to the dots. See also Supplementary Fig. S2. Histograms ofhe FWHM and brightness (mean�S.D.) for different experi-ental conditions are illustrated in Fig. 3 and Fig. S3. Thectual size of AChR nanoclusters was estimated by means ofeconvolution with the effective point spread function in theTED mode.

For a quantitative analysis of the distribution of spatial pointatterns, Ripley’s K-test (Ripley, 1977, 1979) was incorporated. Inarticular, this method allows comparison of observed spatialoint patterns with patterns of complete spatial randomnessCSR) on various length scales by taking into account the dis-ances between all points in an area of interest. The expectedumber of neighbors N(r) for each individual particle within theistance r is characterized by Ripley’s K-function: N(r)�� K(r),here � denotes the particle density of N individual particles in anrea A (��N/A). In the case of CSR, the expected value of N(r) is�r 2 at any distance r, thus K(r) equals �r 2. We calculated K(r)

sing an approximately unbiased estimator as suggested by Rip-ey (1977, 1979):

K�r��N�2A�i�1

N

�j�1, j�1

N

wijk�i, j�

f the distance between the two particles i and j is smaller than r,hen the counter variable k(i,j)�1, otherwise it is zero. The weight-ng factor wij for border correction is 1/(fraction of the circumfer-nce of a circle centered in i and passing through j that lies withinhe analyzed area), yielding values between 1 and 4. Calculation

f k(i,j) and wij from the particles’ coordinates x and y follows from

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R. R. Kellner et al. / Neuroscience 144 (2007) 135–143 137

lementary trigonometry. For interpretation using the transforma-ion L(r)��K�r� ⁄� and calculating L(r)�r is preferred, since(r)�r�0 for random patterns.

The coordinates of the particles were extracted from themages by determining each particle’s center of mass usingustom-written software. The upper and lower value of the 99%onfidence interval (CI) were estimated by means of Montearlo simulations, where for each image the K(r) function for 99

andom patterns of same size and particle density was calcu-ated. To allow averaging of data from different images, thestimated 99% CI values were used to normalize each L(r)unction for each image. Hence the 99% CI is shown as �1 inhe L(r)�r plots.

RESULTS

ide-field fluorescence microscopy images were ob-ained from single membrane sheets prepared by appli-ation of short ultrasound pulses to CHO-K1/A5 cellsrown on glass coverslips. This procedure results in theunroofing” of the cell, leaving only the ventral singlelasma membrane adhered to the surface of the glass.ingle sheets co-stained with the fluorescent probeMA-DPH and Alexa594-�BTX, a quasi-irreversible com-etitive AChR antagonist, are shown in Fig. 1. TMA-DPH

s a general lipid fluorescent probe which enables visu-lization and focusing of thin, two-dimensional speci-ens, i.e. the isolated plasmalemma. TMA-DPH fluores-

ence produces a featureless stain depicting the profilesf a few cell membranes adhered to the glass surface.he corresponding Alexa594-�BTX images reveal anely punctuated appearance of sub-micron sized, dif-

ig. 1. Wide-field fluorescence images of single membrane sheets oby ultrasound treatment as indicated in Experimental Procedures, stay conventional epifluorescence wide-field microscopy; lower panels

raction-limited spots featuring a FWHM of �250 nm. a

Two experimental conditions were used for imaginghole cells and single plasma membrane sheets in bothonfocal and STED microscopy modes. First, fixationas followed by labeling (“fix–label”), in which wholeHO-K1/A5 cells or membrane sheets obtained usingltrasound pulses, were fixed with 4% paraformalde-yde and then labeled with mAb 210 and Atto 532

abeled secondary antibody, thus avoiding long-rangerosslinking of AChRs by the antibodies (Tzartos et al.,987). Second, the labeling procedure preceded fixation“label–fix”) allowing crosslinkage between neighboringChRs. Confocal microscopy revealed fluorescently la-eled dots at the surface of CHO-K1/A5 cells (Fig. 2A,) and sheets (Fig. 4A, B) with an appearance similar to

hose imaged by wide-field epifluorescence in singlelasma membrane sheets (Fig. 1). The average FWHMf the measured spots was 199�34 nm and 191�33 nmhen the specimens were “fix–label” or “label–fix,” re-pectively (Fig. 3).

Applying the doughnut-shaped de-exciting STEDeam (see Suppl. Fig. S1) narrowed the measured spots

o a FWHM of 77.1�32.9 nm and 91.3�40.7 nm for thewo aforementioned experimental conditions, respec-ively (Fig. 3). The striking gain in resolution becomespparent by side-to-side comparison of the high magni-cation confocal and STED microscope images (Figs.B, D and Fig. 4B, D). Since the measured spots rep-esent a convolution of the particles with the finite effec-ive point spread function, the actual protein agglomer-

m CHO-K1/A5 cells. Single plasma membrane sheets were obtainedthe fluorescent cholinergic antagonist Alexa594-�BTX, and examineded-in subfields from their upper counterparts.

tained fro

tions are in fact even smaller. Thus when antibody-

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R. R. Kellner et al. / Neuroscience 144 (2007) 135–143138

nduced clustering is inhibited by fixation, STED imagingesolves the relatively uniform fluorescent spots deliv-red by confocal microscopy into smaller AChR particlesf variable size, with an estimated average diameterelow 55 nm, hereafter referred to as AChR “nanoclus-ers.” This term takes into account the size of fullyeveloped clusters in the adult vertebrate NMJ (re-iewed in Willmann and Fuhrer, 2002; Sanes and Lich-man, 2001) or aneural C2 myotubes (23–94 �m inength, Kummer et al., 2004) and the smallest AChRsub-micron aggregates” visualized by light microscopyn aneural myotubes (Kishi et al., 2005). The histogramsepicting the size and intensity distribution of all spotseasured are shown in Figs. 3 and S3. Table 1 sum-arizes the figures.

Comparing the conditions “fix–label” and “label–fix,”he small increase in the size distribution of AChR nano-lusters reflects the aggregation induced by the com-ined effect of primary (anti-AChR) and secondary (IgG)ntibodies, a combination that results in patching ofell-surface proteins (Prior et al., 2003) and, in our case,n an increase in nanocluster diameter of �60% (Table). Furthermore, a comparison of the size and intensityistribution of AChR nanoclusters in cells (Fig. 2), andpots in single membrane sheets (Fig. 4), revealed al-

ig. 2. AChR nanoclusters can be directly imaged on the cell surfacehow three different cells that were fixed and then labeled with thentibodies. High magnification views of the marked areas provide aD) microscopy. See Figs. 3 and S3 for statistics of size and intensity disith the corresponding distributions as observed by STED microscop

ost no differences for the two conditions tested. This w

nding indicates that the size distribution of AChR par-icles in membrane sheets and whole cells is essentiallyimilar (Table 1).

Cholesterol is one of the key lipid components in-olved in AChR functionality (reviewed in Barrantes,003, 2004). Furthermore, cholesterol-enriched mem-rane domains have been postulated to concentrateignaling molecules and receptors in particular regionsf the cell surface (Maxfield, 2002). We therefore per-ormed a series of experiments, asking whether therganization of AChR particles at the cell surface washolesterol-dependent. Acute CDx treatment is an es-ablished procedure to deplete cholesterol from the plas-alemma (see review by Pichler and Riezman, 2004)nd to study the effect of cholesterol on the clusterrganization of membrane proteins (e.g. Prior et al.,003). To study the effect of cholesterol depletion fromHO-K1/A5 cells, these cells, or single membraneheets, were treated with 10 mM CDx at 37 °C for 20in, labeled with the monoclonal antibody mAb 210,irected against the main immunogenic region of theChR protein (Tzartos et al., 1987) followed by the Atto32-labeled secondary antibody, and then fixed (condi-

ion “CDx–label–fix”). Using this experimental conditionn single plasma membrane sheets, epifluorescence

t CHO-K1/A5 cells. The low magnification 20�20 �m2 images (A, C)0 monoclonal anti-AChR antibody and Atto 532-labeled secondaryde comparison of the resolution achieved in confocal (B) and STEDof the fluorescent AChR spots in confocal microscopy and comparison

of intacmAb21

side-to-si

ide-field microscopy studies performed in a previous

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R. R. Kellner et al. / Neuroscience 144 (2007) 135–143 139

ork revealed an increase in the intensity of the fluores-ent spots, which we interpreted as a consequence ofhe antibody-induced tethering of AChR particles uponDx treatment (Borroni et al., in press). However, weould not resolve any changes in particle size. Similarly,o statistically significant difference in spot size could bebserved between control and CDx-treated samples byonfocal microscopy (e.g. Fig. 3). Once again, changesere only apparent in the intensity distribution of thepots which were brighter upon cholesterol depletionsee Table 1 and Suppl. Fig. S3). Whether cholesterolepletion facilitated AChR supramolecular aggregationemained an open question requiring higher spatial res-lution to be responded.

As shown in Figs. 5, 3 and Table 1, STED micros-opy revealed changes in the distribution of AChR par-icles upon CDx treatment that were not apparent inonfocal microscopy. The analysis of the size and fluo-escence intensity distribution of AChR particles in theTED images exhibited a significant broadening of theize distribution. This broadening stems from furtherggregation of the small AChR particles, resulting in

arger-sized AChR nanoclusters, as detected by STEDicroscopy, and increased spot intensity as also ob-

erved by wide-field (Borroni et al., in press) and con-ocal microscopies (Fig. S3).

The distribution of AChR clusters in the fluorescencemages was also analyzed at larger scales by applyingipley’s K-function (Ripley, 1977, 1979). Representing aecond-order analysis of spatial point patterns, the K-func-

0 50 100 150 250 300 350

20

40

60

80

100

20

40

60

80

100

0 50 100 150 200 250 300 350

50

100

150

200

250

300

0 50 10

50

100

150

200

Con

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Mean Spot Size77.1 ± 32.9 nm

ig. 3. Size distribution of AChR spots in confocal microscopy and ACefer to the three experimental conditions tested in this work as inxperiments. The total number of particles analyzed is indicated in Tab

n the STED images than in the confocal images. Deconvolution of the m99.9% upper limit estimate for the mean AChR particle size of 55 n

CDx–label–fix”).

ion is used to test for spatial randomness. As opposed to l

earest neighbor methods, all inter-particle distances overhe study area are incorporated into the analysis, thusroviding a thorough topographical characterization which

n turn is compared with that of a pattern resembling CSRRipley, 1977, 1979; see Experimental Procedures andupplementary data). The data corresponding to wholeells in “label–fix” condition and imaged with STED micros-opy showed a random distribution of particles at all lengthcales (Fig. 6A), i.e. the deviation from the L(r)�r�0 curveas within the 99% CI expected for a random distribution.hen the same analysis was performed on the membrane

heets (Fig. 6B), the calculated L(r)�r metrics of controllasma membrane sheets and intact cells showed theame tendency.

Depletion of cell-surface cholesterol (Fig. 5) pro-uced a marked alteration of the long-range organiza-

ion of the AChR in whole cells (Fig. 6A). After choles-erol extraction from the CHO-K1/A5 cells, positive val-es of the L(r)�r function were observed within a narrowegion of the r range analyzed; maximum deviation fromhe L(r)�r�0 curve occurred at radii of �0.5–1 �m,apidly falling off to values below the cutoff limit fornter-cluster distances 1.5 �m. Thus cholesterol de-letion is accompanied by an increase in long-range

nteractions (as compared with the nanometer scale ofhe AChR clusters themselves) and hence a change inChR cluster distribution made apparent by STED mi-roscopy. The clustering of particles was clearly ob-erved in intact cells but was barely apparent in singleembrane sheets, where it could be observed only for

0 50 100 150 200 250 300 350

20

40

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100

200 250 300 350 0 50 100 150 200 250 300 350

50

100

150

200

ze [nm]

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200 250 300 350

Spot Size 40.7 nm

Mean Spot Size125.0 ± 52.6 nm

les in nanoclusters resolved by STED microscopy. The three columnsnd the histograms represent averages of five independent sets ofdeterminable mean spot size (vertical red lines) is noticeably smallerspots with the effective point spread function of the STED mode gives

ition “fix–label”), 69 nm (condition “label–fix”) and 116 nm (condition

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R. R. Kellner et al. / Neuroscience 144 (2007) 135–143140

DISCUSSIONsing STED microscopy we were able to study changes in

he size distribution of AChR nanoclusters well below theiffraction limit of a typical wide-field and a confocalicroscope.

The findings of the size distribution analysis indicate thathe organization of AChR nanoclusters at the plasma lemmas indeed non-random, as well as cholesterol-dependent atcales below 250 nm. In an elegant recent study using total

ig. 4. AChR spots and AChR nanoclusters in single plasma membrpplied to CHO-K1/A5 cells adhered to the glass coverslip, fixed, labele

he entire glass-adhered plasma membrane. Right column: high magonfocal spots into multiple AChR nanoclusters with STED microscop

able 1. Size and fluorescence intensity distribution of AChR nanoag

ondition Spot size (nm) Estim

Mean S.D. Mean

hole cells

Fix–label” 75.8 32.4 38.8Label–fix” 109.7 35.7 92.1CDx–label–fix” 120.0 44.74 105.0

embrane sheets

Fix–label” 77.1 32.9 41.1Label–fix” 91.3 40.7 63.3CDx–label–fix” 131.1 60.1 118.8

The figure in parentheses (n) indicates the number of AChR particles

f simulated clusters with the measured point spread function.

nternal reflection fluorescence (TIRF) microscopy (Demurond Parker, 2005), AChRs heterologously expressed in oo-ytes appear to exhibit a random distribution when indirectly

maged by fluorescence changes in Ca2� fluxes. The ran-om, non-clustered distribution of AChRs in the oocyte mem-rane could be related to the fact that non-aggregating, tran-iently expressed embryonic-type (�2��) AChRs were im-ged in the recent Demuro and Parker (2005) study, with a

ateral resolution of �1 �m. By contrast, in our study we

ts. Plasma membrane sheets were obtained by an ultrasound pulsen imaged in the confocal (A, B) and STED (C, D) modes. Left column:detail of a 1.95�1.95 �m2 area illustrating the resolution of single

as determined by STED microscopy

ter size (nm) Intensity (a.u.)

99.9% Upper limit Mean S.D.

53.8 347.7 266.5 (n�609)95.3 955.5 868.5 (n�541)

110.1 797.3 692.5 (n�734)

55.1 323.4 302.5 (n�1075)69.4 442.2 409.4 (n�817)

123.1 701.3 874.4 (n�616)

in each case. The cluster size was estimated by means of convolution

ane sheed and the

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R. R. Kellner et al. / Neuroscience 144 (2007) 135–143 141

mploy a clonal cell line stably expressing adult-type (�2���)ChR, an interesting difference worth addressing in futuretudies. More likely, however, the disclosure of the clusteredupramolecular organization of AChRs in the membrane, andts cholesterol dependence, are a further indication of thebility of STED microscopy to reach hitherto unexplored nan-dimensions in biological imaging and provide new informa-ion on the supramolecular architecture of the AChR.

The results of the spatial point pattern analysis revealedhat the long-range AChR organization at the plasmalemmaf CHO-K1/A5 cells depends on cholesterol-sensitive inter-ctions that normally extend over the range of a few microns

n untreated cells (�1 �m under our image sampling size, cf.ig. 6A). A likely candidate for the maintenance of such an

nfluence is the cortical cytoskeleton and, particularly, thectin network (e.g. Kwik et al., 2003). The ability of AChRanoclusters to aggregate upon cholesterol depletion inhole cells (Fig. 6A) is apparently lost or substantially atten-ated in single plasma membrane sheets devoid of the sub-ortical cytoskeleton (Fig. 6B). This provides additional, albeit

ndirect, evidence that the long-range AChR supramolecularrganization is likely to be associated with the presence of an

ntact cytoskeletal network under physiological energy supply

ig. 5. STED microscopy reveals differences in AChR organizationanoclusters imaged by STED microscopy of single plasma membr.5�7.5 �m2 in size. Cholesterol depletion (10 mM CDx, 30 min, 37ompared with controls (A, B). The size distribution also changes, aendency toward larger size of AChR nanoclusters upon cholesterol dep

nd normal cholesterol levels (Borroni et al., in press). In B

uscle, AChRs have been reported to be associated withctin via urotrophin (Willmann and Fuhrer, 2002). The short-ange extent and composition of the AChR nanoclusters isrobably maintained by protein–protein (i.e. receptor–recep-

or) and receptor–lipid interactions, of which AChR–choles-erol interactions may constitute the prevailing stabilizingorce (Barrantes, 2004).

The present study is the first application of nanoresolv-ng light microscopy to the study of the spatial distributionf an ion channel. Since AChR constitutes the paradigmithin the large family of rapid ligand-gated ion channels,

he ability to spatially map its supramolecular distributions well as to determine the size of its nanoaggregatespens new avenues for harnessing the concept of STEDuorescence microscopy to the study of myriad mem-rane proteins. Moreover, as it is inherently unlimited byiffraction, improvements on the STED technique are

ikely to increase its usefulness in the neurosciences.

cknowledgments—This work was partly supported by a jointrant from Fundacion Antorchas (Argentina)/DAAD (Germany) to.J.B. and S.W.H. F.J.B. acknowledges partial support fromONICET, FONCyT and Univ. Nac. Sur. Thanks are due to Ms

about by cholesterol depletion from the plasma membrane. AChRts from CHO-K1/A5 cells: A and C show low magnification views,lts in the diminution of the number of AChR nanoclusters (C, D) asappreciated in the high magnification images (B, D), with a markedSee corresponding histograms in Figs. 3 and S3, and also Table 1.

broughtane shee°C) resu

eatriz de los Santos for help in cell culture, to Jan Keller for

dW

A

B

B

B

Fbpcma

R. R. Kellner et al. / Neuroscience 144 (2007) 135–143142

oughnut optimizations, and to Andreas Schönle and Volkerestphal for providing software for image analysis.

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APPENDIX

upplementary data

upplementary data associated with this article can be found, in

he online version, at doi: 10.1016/j.neuroscience.2006.08.071.

(Accepted 29 August 2006)(Available online 16 October 2006)