Visualization of single proteins from stripped native cell membranes: A protocol for high-resolution...

Visualization of Single Proteins From Stripped NativeCell Membranes: A Protocol for High-ResolutionAtomic Force MicroscopyCARLOTTA MARASINI,1* EMANUELA JACCHETTI,1,2 MANOLA MORETTI,3 CLAUDIO CANALE,3

OSCAR MORAN,1 AND MASSIMO VASSALLI1

1Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, 16149, Italy2NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Pisa, 56127, Italy3Nanophysics Dipartimento, Istituto Italiano di Tecnologia, Morego, Genova, 16163, Italy

KEY WORDS AFM; CFTR; fluorescence; single molecule

ABSTRACT Atomic force microscopy (AFM) proved to be able to obtain high-resolutionthree-dimensional images of single-membrane proteins, isolated, crystallized, or included inreconstructed model membranes. The extension of this technique to native systems, such as theprotein immersed in a cell membrane, needs a careful manipulation of the biological sample tomeet the experimental constraints for high-resolution AFM imaging. In this article, a generalprotocol for sample preparation is presented, based on the mechanical stretch of the cell mem-brane. The effectiveness for AFM imaging has been tested on the basis of an integrated opticaland AFM approach and the proposed method has been applied to cells expressing cystic fibrosistransmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possi-bility to identify and analyze single proteins in the plasma membrane. Microsc. Res. Tech.76:723–732, 2013. VC 2013 Wiley Periodicals, Inc.

INTRODUCTION

Atomic force microscopy (AFM) is nowadays exten-sively used for the characterization of biological sys-tems at the nanoscale and its applications span fromwhole cells down to single fibrils or proteins (Garciaand Herruzo, 2012; Le Grimellec, 1998; Papi, 2010). Inparticular, the possibility to obtain high-resolutionthree-dimensional (3D) reconstructions of single mole-cules in quasi-physiological environment is one of thekey aims of such a microscopy technique. So far, singlemolecules have already been imaged by AFM (Fotia-dis, 2012; Fotiadis et al., 2002), working on purifiedproteins anchored to hard surfaces (typically flat micasheets) (Dante et al., 2011; Frederix et al., 2009; Kise-lyova et al., 1999; Muller, 1997, 2008; Sbrana et al.,2007), by fostering the formation of two-dimensionalcrystals (Moller et a., 2003; Stahlberg et al., 2001), orimaging on a reconstructed membrane (Monaldi et al.,2010; Muller, 2008) but there is a growing interest inextending this capability from the model toward nativesystems. Cell surface investigation by AFM is integra-tive to biochemical, fluorescence, and electron micros-copy methods, and can elucidate the structuralproperties of proteins on cell membranes, which are ofparamount importance on the cell physiology. Manyhuman disorders are associated to membrane proteinmis-functioning or destructuring and important scien-tific questions will benefit of an experimental tech-nique able to directly observe single molecules, in theirfunctional environment. In such a perspective, AFMcould play a crucial role but its ability to disclose theconformation of membrane proteins in native environ-ment is strongly influenced by the application of a

reliable sample preparation protocol. To exploit theAFM resolution capabilities, it is mandatory to have aclean, flat, and mechanically stable surface to work on.So far, an operating protocol to isolate and study trans-membrane proteins was proposed for specialized sys-tems, such as red blood cells and frog oocytes (Orsiniet al., 2010; Schillers et al., 2001, 2004), and someattempts have also been made for more general pur-pose eukaryotic cells (Wang et al., 2010), but a definiteprotocol to attain single-molecule visualization innative plasma membrane has not yet been established.

The aim of this study was to identify a method to iso-late membranes from living cells, so as to obtain an opti-mal sample for high-resolution AFM investigation ofsingle proteins. A panel of experimental tests wasadopted to assess the preparation protocol effectiveness,using a combined approach based on bright-field opti-cal, fluorescence, and AFM. The method was tested on arelevant biological system, the cystic fibrosis transmem-brane conductance regulator (CFTR), a chloride chan-nel whose dysfunction causes cystic fibrosis, one of themore common recessive Mendelian genetic diseases inthe world (Riordan et al., 1989). This is an outstandingexample in which a full knowledge of the CFTR struc-ture and conformation would allow a better

*Correspondence to: Istituto di Biofisica, Consiglio Nazionale delle Ricerche,Via De Marini 6, Genova 16149, Italy. E-mail: [email protected]

Received 26 March 2013; accepted in revised form 8 April 2013

Contract grant sponsor: Fondazione per la ricerca sulla fibrosi cistica;Contract grant number: FFC#6/2009; Contract grant sponsor: FFC delegation“Lago di Garda”, support groups of Chivasso and Isola bergamasca.

DOI 10.1002/jemt.22223Published online 16 May 2013 in Wiley Online Library (wileyonlinelibrary.com).

VVC 2013 WILEY PERIODICALS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 76:723–732 (2013)

understanding of the disease, potentially leading to thedesign of suitable drugs (Galietta and Moran, 2004).

MATERIALS AND METHODSCell Culture

Fisher rat thyroid (FRT) cell line native and stablytransfected with a cDNA coding for wild-type CFTRchannel were used in the experiments. Cells were grownin Coon’s medium complemented with 10% fetal bovineserum (FBS), 2 mM glutamine, and maintained at 37�Cand 5% CO2. Except when indicated, all reagents werepurchased from Sigma-Aldrich (St. Louis, MO).

Sample Preparations

Muscovite mica, culture plastic Petri dishes, andglass coverslips were used as sample substrate formembrane preparation. Sterility and absence of dustand contaminants were assured on all substrates.Mica was freshly cleaved under sterile head beforeuse; plastic sterile petri dishes were used as-is; glasscoverslips were washed with a 1:1 methanol and hy-drochloric acid solution and thus autoclaved.

A variable volume (50–300 ll) of 1–8 3 105 FRT cellsin suspension was seeded on mica, glass, or plasticsubstrates, with and without poly-L-lysine coating, andincubated from 20 min to 5 h in serum-free culture

medium. After washing, the solution was removed andthe sample was reversed and put on top of a clean cov-erslip, sandwiching the cells between the two surfaces.To increase the pressure, weighting pieces (about 20 g)were put on the sandwich for a waiting time rangingfrom 5 to 60 min. Afterward, the sandwich wasstripped, taking the lower coverslip as the imagingsample (not the substrate used for cells seeding). Fig-ure 1 shows this mechanical procedure. Particularcare should be put in washing the sample, to obtainclean membranes, minimizing the cytoplasmic organ-elles presence. Membranes were washed four times inTris buffer solution (TBS, 150 mM NaCl, 10 mM Tris,pH 8.0) and twice with filtered deionized water. Toincrease the stability, the sample was fixed overnightwith 4% formaldehyde solution in phosphate-bufferedsaline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mMNa2HPO4, 1.46 mM KH2PO4, pH 7.4).

Optical and Confocal imaging

Optical images were obtained with a Zeiss Axiovertoptical microscope (Zeiss, Germany), whereas confocalimages were obtained using three-channel Leica TCSSP5 (Leica, Germany) laser-scanning confocal micro-scope, equipped with single- and two-photon excitation,through a plan-apochromatic oil immersion objective

Fig. 1. Schematic representation of the technique used to isolateplasma membranes, based on a mechanical stripping procedures.Cells are seeded and grown on an appropriate substrate (indicated asseeding substrate (A)). A sandwich is created with a second a cleancoverslip, reversed, and left stabilizing in humid but not liquid envi-ronment for some time (for details, see the text). To increase the

pressure in this phase, some weigh pieces are put on top of the sand-wich (B). Finally, the mechanical stripping is performed and the sec-ondary coverslip (not the seeding substrate) is taken as the imagingsample (C). [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

724 C. MARASINI ET AL.

Microscopy Research and Technique

(633/1.4). Membranes were labeled using an amphi-philic fluorescent styryl pyridinium dye specific for lip-ids (FM4-64, Molecular Probes Europe, Leiden, TheNetherlands); actin cytoskeleton was labeled usingTRITC-conjugated phalloidin (Life Technologies, Italy)and cell nuclei were stained with 40,6-diamidino-2-phe-nylindole (DAPI) (Molecular Probes, Europe). Excita-tion wavelengths were selected to minimize channelscrosstalk: 750 nm two photon lasers for DAPI; 488 nmfor FM4-64 and 543 nm for TRITC fluorescent protein.Although the 488-nm line excites only FM4-64, the 543-nm laser results in a merge between FM4-64 andTRITC signals. A linear unmixing procedure based on acrosscorrelation analysis was performed to isolate theTRITC signal on the second channel (Kraus et al., 2007).

Immunogold Labeling

Cellular membranes were stained with gold-conju-gated antibodies. Nonspecific antibody bindings wereblocked with 4% of bovine serum albumin (BSA) inPBS for 2 h. The samples were treated with the pri-mary antibody specific for the cytoplasmic side ofCFTR (MM13-4, Millipore, Billerica, MA) (Claasset al., 2000; Mendes et al., 2004). The primary anti-body, in a dilution of 1:100 in 1% BSA in PBS, wasincubated with the sample for 2 h at room tempera-ture. After washing with filtered PBS, membraneswere incubated for 2 h at room temperature with gold-conjugated goat antimouse immunoglobulin (Frankelet al., 2006; Schillers et al., 2004), diluted at 0.5% in asolution containing 0.5 M of NaCl, 0.1% BSA, and 5%FBS. The secondary antibody was labeled with 10-nmdiameter gold spheres.

AFM Imaging

AFM measures were carried out using a NanowizardII by JPK (Berlin, Germany) mounted on top of a ZeissAxiovert inverted optical microscope. The coupling withthe optical microscope allows for the integration of high-quality optical imaging, fluorescence imaging, and AFMimaging. AFM was operated both in contact mode andin tapping mode, in air on dried samples and in liquidenvironment (PBS). The AFM cantilevers were selectedrespective to the working mode: CSG11 (NT-MDT, Mos-cow, Russia) for contact mode, NSG10 (NT-MDT, Mos-cow, Russia), and SS-NCH ultra-sharp (Nanosensor,Neuchatel, Switzerland) for noncontact mode. Imagingefficiency in dry and liquid environment on fixed sam-ples was tested on several samples, showing no statisti-cally relevant difference (data not shown) and hereafterimages acquired only in dry environment are shown.

A custom coverslip holder was realized to maximizethe accessible area of the sample, allowing to searchfor regions of interest with the optical microscope andthus to focus on selected features with the AFM. Theexact superimposition of the corresponding imageswas obtained by using the DirectOverlay routine of theAFM acquisition software (JPK Instruments, Berlin,Germany).

RESULTSSample Preparation Methods

Several techniques, based on already developed lit-erature protocols, were tested to obtain suitable

samples to study single-membrane proteins with AFM.For this purpose, plasma membranes have to be iso-lated, cleaned (to prevent the presence of any residualcytoskeleton structure that could prevent the access tothe membrane itself), and mechanically stable(attached to the substrate). In addition, the coverslips,where the sample was fixed, must be flat, rigid, andtransparent to combine the optical and AFM meas-ures. All approaches tested required four steps: (1)cells growth over the selected substrate, (2) breakingof the cells, (3) washing and cleaning of isolated mem-branes, and (4) membrane fixation over the imagingsubstrate. To optimize the sample preparation, severalparameters were changed: the cell density (30,000–300,000 cells/cm2), the incubation time (30 min–24 h)to modulate the cell adhesion to the coverslips, thesubstrate (mica, glass, plastic; with and without poly-L-lysine [PLL]), and the method used to burst the cells.This final step resulted to be the crucial point to obtainoptimal substrates.

Several approaches were proposed in the literatureto obtain a reliable breaking of all population. The ma-jority of the authors proposed a method based on thedirect mechanical stripping of the cells (Cohen et al.,1977; Frankel et al., 2006; Ehrenhofer et al., 1997) butsome trials were also done by means of an ice-cold-based protocol (Bezrukov et al., 2009). Bothapproaches were tested and several variations of ex-perimental parameters were evaluated. In particular,for ice-cold method, the cells were washed with ice-cold water and incubated in ice for a variable time (20min–3 h). Regarding the mechanical stripping proto-col, detailed in the Materials and Methods section,particular care was also taken for stabilizing weightselection (2–30 g).

Each experimental protocol, either mechanical strip-ping or ice-cold breaking, was tested against differentsubstrate: mica, glass, and plastic. The effectiveness ofeach approach was verified via a panel of integratedmicroscopy techniques detailed in the followingsection.

Optical Analysis

The first step used to estimate the preparation qual-ity and to evaluate whether clean membrane regionsare present was the optical characterization of thesample.

The difference between a sample prepared with themechanical stripping method and the intact cells (cellsseeded and directly fixed) resulted evident at a glance(Figs. 2A and 2B). In the case of the stripped sample,cells appeared thinner and less rounded than intactcells. Some biological material was dispersed on thesubstrate. An image at high magnification of the sam-ple allowed to distinguish between regions with arough morphology (Fig. 2C), which must be avoided inAFM measures, and zones with a completely differentmorphology that can be observed only in phase con-trast (Fig. 2D). Spots of interest, with clean isolatedmembranes, were found in regions of the latter type,appearing thinner at an optical inspection.

To corroborate this optical inspection, that allows tohighlight suitable zones for AFM measures in a sam-ple, fluorescence microscopy was additionally per-formed (Fig. 3). Samples treated with fluorescent dyes

AFM ON SINGLE MEMBRANE PROTEINS 725


for membranes, cytoskeletal structures, and nucleiwere investigated by confocal laser scanning micros-copy. As expected, samples with intact cells (Fig. 3A)were characterized by the presence at more nuclei(stained with DAPI, colored on blue). Moreover, suchsamples showed only zones with the colocalization ofthe signal of FM4-64 and of phalloidin–TRICT, indicat-ing regions where the cytoskeleton structures werestill anchored to the plasma membrane (colored in yel-low). Zones with only clean membranes were neverfound.

In the fluorescence image of the samples preparedwith the ice-cold breaking technique, (Fig. 3B), it waspossible to see some regions with the signal of FM4-64alone, reddish regions, indicating isolated membranes.Despite this fact, many regions with evident cytos-keletal structures were present in ice-cold-treatedsamples and some nuclei were even still present.Owing to its poor yield, this technique was discarded.

Conversely, samples managed with the mechanicalstripping method showed very different results. Noblue-stained areas were ever present (Fig. 3C), indicat-ing that cells were no longer intact and nuclei hadbeen washed out. Some regions still showed the super-position between TRICT and FM4-64 emission (greenarrows), but more zones associated to clean membraneareas (red arrows) were shown. In addition, using the3D reconstruction feature of the Leica SP5 confocalmicroscope, it was observed that yellow regions wereup to 1 lm thick, spanning many 3D slices, whereasred-only regions were thinner (data not shown).

AFM Inspection

A more detailed inspection of the samples was per-formed by AFM imaging in contact mode on dried sam-ples. Being able to provide quantitative information onthe morphology, this is the ultimate technique to

Fig. 2. A: Optical image of intact cells seeded on a glass coverslip(magnification, 403; air objective). B: Image of a treated sample in aregion in which cells are no longer present but the biological materialhas a rough morphology that preludes to a sample in which cleanmembranes can not be found (magnification, 403; air objective). C,

D: Zoom of regions of interest acquired with a 1003 oil immersionobjective in phase contrast mode that show a morphology ideal forfurther AFM inspection. [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]



assess the effectiveness of the sample preparation. Anisolated clean plasma membrane should look like aplane sheet of about 5–10 nm height with a roughnessassociated to the height of membrane proteins. Indeed,regions in which the cleaning was not optimal and con-taminations (from cytoskeleton or other intracellularorganelles) were still present are clearly evident asthey present brighter spots with profiles higher thanthe expected 10 nm. To have a reference, intact cells,before any breaking treatment, were observed withAFM, in dry environment, just after fixation on a cov-erslip. Figure 4 shows a reference result in which typi-cal width (ca. 20 lm) and height (ca. 1 lm) arequantified.

The same analysis was also performed on the sam-ples prepared with the mechanical stripping method.In this case, the measure of the height of the severalzones of the sample allowed to distinguish betweenthicker (cytoskeleton structures still anchored to themembrane) and thinner regions (clean membrane).Figure 4 shows an example from selected samples inwhich many biological unwanted structures remainedanchored to the membranes, with a huge morphology.Isolated clean membranes could be found performing ahigh-magnification image in regions looking flatter atoptical inspection (see the previous section) and itcould be fully characterized by AFM. Figure 5A showsan example of a region in which a large (few micro-meters) clean membrane sheet was present. A refer-ence profile is plotted in Figure 5B and a statisticallyrelevant evaluation of the step height can be obtainedfrom the distance between the peaks in the height his-togram (Fig. 5C). Upon fitting the histogram with twoGaussian profiles, a measure of �5 nm for the showedimage was obtained.

Effect of Poly-L-lysine

To increase the efficiency of the mechanical strip-ping preparation method, a PLL (Sigma-Aldrich St.Louis, MO) coating coverslip was used to enhance thecell adhesion on the selected substrate. Unexpectedly,

AFM inspection of PLL-coated substrates often showedparticular annular structures as shown in Figure 6A,which could not be related to any recognizable cellularfeature. For the sake of clarity, bare PLL-coated sub-strates were investigated before all seeding. Figure 6Bshows a reference image, clearly showing similar an-nular structures. In addition, PLL created some arti-facts. Figure 7 shows isolated membranes on a treatedglass substrate that appeared as a good preparation.The membrane sheet, in this case, seems to be foldedon itself and some steps are identified in the imageshown in Figure 7. This peculiar behavior is high-lighted in the histogram of Figure 7C, in which four,almost equally spaced, peaks are clearly visible. Thisstructure can be easily associated with folded mem-brane sheets, also because the step height is compara-ble with a phospholipid bilayer, but it was observedonly in the presence of PLL and should be treated asan artifact. For this reason, PLL coating was avoidedin the final preparation protocol.

Immunogold Labeling for Single-ChannelVisualization

To have a complete assessment of the preparationprocedure, the sample was selectively stained with anindirect immunohistochemistry of 10-nm gold spherestargeted to the cytoplasmic side of CFTR channels.This procedure is suitable to verify the placement ofthe membranes on the substrate and to recognize theintracellular side of the protein. The specificity of thelabeling was tested on membranes extracted from FRTcells, which endogenously do not express the CFTRchannel. In the case of wild-type FRT samples, treatedwith gold-conjugated antibody, spherical structuresclearly associated to gold particles were never found instandard conditions and the morphology was not dif-ferent from that of observed in untreated cells (datanot shown).

On the contrary, samples prepared from FRT cellsoverexpressing CFTR presented rounded particles,with a size comparable to the gold spheres nominal

Fig. 3. Confocal fluorescence microscopy images samples treated indifferent ways: (A) intact cells, (B) ice-cold stripped cells, and (C)mechanically broken cells. All the samples are treated to highlightthe nucleus (DAPI, blue), actin filaments (TRICT–phalloidin, green),and membranes (FM4-64, red). Images are acquired with a plan-apo-chromatic oil immersion objective (633/1.4). White bars represent 20

lm. The fact that regions of the sample appear as yellow is owing tothe superposition of TRICT and FM4-64 emission signals. Arrows in(C) point out regions with putatively clean membrane (red arrows)and regions in which actin fibers are still present (green arrows); fora detailed description, see text. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]



diameter (Fig. 8, arrows in the inset). The resolution ofthe imaging system can thus be stressed, (Fig. 9) tovisualize the conformation details of single particles inplasma membrane.

DISCUSSION

A general procedure to assess the effectiveness ofthe membrane isolation and fixation technique wasdeveloped and applied to identify a reliable and repro-ducible protocol. Several approaches were testedagainst the presented validation procedure, using acombination of optical bright field (Fig. 1), fluorescence(Fig. 3), and AFM (Fig. 4), allowing for a fine charac-terization of the selected samples (Fig. 5).

The crucial step in the described protocol is themethod used to burst the cells. An approach based onthe use of ice-cold solutions to break the cells after in-hibiting the volume regulation mechanism was tested,based on the previously proposed description for Xeno-pus oocyte (Schillers, 2008; Schillers et al., 2001, 2004)and mammalian fibroblast (Bezrukov et al., 2009).However, these approaches were not effective on FRTcells, more stable than the cellular systems previouslyused. A more general approach was thus adopted,based on the mechanical stripping of cells sandwiched

between two layers (Cohen et al., 1977; Ehrenhoferet al., 1997) (Fig. 1). After trying several conditions,the best result was obtained with 10 min of pressingtime and under a homogeneously distributed weight of20 g. The mechanical stripping procedure needs to beperformed in wet environment (but not in liquid asproposed by Ehrenhofer et al., 1997), and using glasscoverslips rather than treated mica (Cohen et al.,1977) for both sandwich sides. The seeded cells have tobe at subconfluent density (ca. 120,000 cells seeded in150 lL for each coverslip, 1 cm of diameter, for 3–4 h).Cells fixation before the stretching (Ehrenhofer et al.,1997), instead, does not provide significant enhance-ments to obtain membrane monolayer and it is thusdiscouraged. Moreover, the onset of possible artifactsassociated to the PLL coating is shown (Figs. 6 and 7)and suggests to use only raw glass coverslips.

Besides the proposed cell membrane preparationprotocol, a simple and effective method to target sin-gle-protein molecules on clean membrane patches isapplied. Stripped cells were fixed with formaldehyde tostabilize the sample for AFM inspection in contactmode, and treated with the lipophilic dye FM4-64 tohighlight isolated membranes by fluorescence micros-copy. Interestingly, this dye is fluorescent also in dry

Fig. 4. A: Reference AFM image of an intact cell from a nontreatedsample. The image was acquired in contact mode in air environmentand it shows a morphology with a typical height of about 1 lm (seeprofile in (B) indicated by the white line). (C) Reference AFM image,obtained in contact mode in air environment, of a relevant structuretaken from a mechanically stripped sample. Cells are no longer intact

and the whole typical morphology is lost, but rough structures, asso-ciated to cellular biological material of about the same height (seeprofile in panel D), are still present. Scale bars: (A) bar, 20 lm; totalvertical range, 1.4 lm; (C) bar, 5 lm; total vertical range, 1.35 lm.[Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]



environment, allowing to perform high-resolutionAFM imaging in dry conditions, without loosing therecognition capabilities provided by the fluorescencesignal. As a reference, Figure 10A shows the fluores-cence image obtained on a sample in which the bright-est spots correspond to higher regions, where clean

membranes are not expected to be found, as verifiedupon AFM analysis in regions 1 and 2. Moreover, thefluorescence signal allowed to discover some regions,invisible in standard optical imaging, such as the spotin region 3 of Figure 10, where clean membranes werepresent. The targeting of these zones allowed to

Fig. 5. (A) AFM image of a clean plasma membrane sheet obtained in contact mode in air environ-ment. (B) Height profile taken from the image in (A). (C) Histogram of the height distribution of panel(A). Scale bars: (A) bar, 10 lm; total vertical range, 167 nm. [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

Fig. 6. Two representative AFM images, obtained in contact modein air environment, of typical annular structures associated to thePLL polymerization. (A) Picture obtained on a substrate on which cellwere seeded. (B) High-resolution zoom on a bare PLL-coated

substrate. Scale bars: (A) bar, 20 lm; total vertical range, 356 nm; (B)bar, 400 nm; total vertical range, 18 nm. [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]



Fig. 7. A: AFM image, acquired in contact mode in air environ-ment, of a huge artifact caused by PLL on a stripped cell sample. B:Height profile taken from the white line in (A). Red line is a guide forthe eyes, highlighting the presence of defined steps. C: Height

distribution histogram of (A) with a multi-Gaussian fit (dashed line).Scale bars: (A) bar, 20 lm; total vertical range, 500 nm. [Color figurecan be viewed in the online issue, which is available atwileyonlinelibrary.com.]

Fig. 8. AFM image, obtained in contact mode in air environment, ofa clean plasma membrane labeled with anti CFTR-antibody conju-gated with 10-nm gold spheres. The inset in the squared region isacquired at higher resolution to highlight the presence of the goldspheres. Scale bars: (image), bar 1 lm; total vertical range, 56 nm;(inset) bar, 30 nm; total vertical range, 32 nm. [Color figure can beviewed in the online issue, which is available atwileyonlinelibrary.com.]

Fig. 9. High-resolution AFM image, acquired in contact mode in airenvironment, on a sample with immunogold-labeled clean membraneextracted from FRT cells expressing CFTR. By zooming and stressingthe resolution nearby a gold sphere (squared inset), it is possible toobserve the conformation of a single channel. Scale bars: (both imageand inset) bar, 100 nm; total vertical range, 22 nm. [Color figure canbe viewed in the online issue, which is available atwileyonlinelibrary.com.]



measure single-membrane proteins with high-resolu-tion AFM, like the zoomed image of Figure 8. The pro-posed approach toward high-resolution imaging ofsingle proteins in native membranes was tested on acell line overexpressing CFTR, identified by an immu-nogold labeling technique (Figs. 8 and 9). Zooming intoclean membrane patches and concentrating the inspec-tion nearby gold spheres can lead to the individuationof annular structures (Fig. 9) about 9 nm thick andwith inner hole diameter of roughly 25 nm. These find-ings are in complete accordance with the previousresults on the same channel protein (Schillers, 2008),obtained on membrane patches extracted from frogoocytes and red blood cells.

CONCLUSIONS

The optimal preparation of clean cellular mem-branes was addressed to gain mechanical access to thecytosolic side and to perform high-resolution AFMimaging of a transmembrane protein, namely CFTR.The sample preparation procedure was deeply testedto attain a stable and reliable membrane extractionand attachment protocol. Optical microscopes wereable to roughly discriminate samples suitable (or not)for further AFM measurements. By using AFM, it isalso possible to highlight potential preparation arti-facts, such as big annular structures or thin step-likestacking of sheets that are likely to give no contribu-tion to further AFM analysis. The feasibility of single-molecule AFM recognition and imaging on nativeplasma membranes was verified by immunogold stain-ing of CFTR, showing the ability to resolve nanometricstructures comparable to those observed in the litera-ture on a Xenopus oocyte (Schillers, 2008).

In conclusion, this study developed a protocol toobtain clean plasma membrane from mammalian cells.This technique allows to study single molecules intheir natural environment at high resolution with adirect technique such as AFM and it will allow to

analyze not only the structure but also the functionalproperties of these molecules. This is, indeed, a rele-vant result because some membrane proteins areinvolved in important diseases, as, for instance, CFTRfor cystic fibrosis, and with this method it will be possi-ble to gain crucial structural and conformational infor-mation in native environment.

ACKNOWLEDGMENTS

The authors thank Mattia Pesce, Cesare Usai, andMichele Ferrari for their support in imaging measuresand Olga Zegarra-Moran and Gino Galietta for usefulsuggestions and the provisioning of FRT cell lines.

REFERENCES

Bezrukov L, Blank PS, Polozov IV, Zimmerberg J. 2009. An adhesion-based method for plasma membrane isolation: Evaluating choles-terol extraction from cells and their membranes. Anal Biochem394:171–176.

Claass A, Sommer M, de Jonge H, K€alin N, T€ummler B. 2000. Applic-ability of different antibodies for immunohistochemical localizationof CFTR in sweat glands from healthy controls and from patientswith cystic fibrosis. J Histochem Cytochem 48:831–837.

Cohen C, Kalish D, Jacobson BS, Branton D. 1977. Membrane isola-tion on polylysine-coated beads: Plasma membrane from HeLacells. J Cell Biol 75:119–134.

Dante S, Haub T, Steitz R, Canale C, Dencher NA. 2011. Nanoscalestructural and mechanical effects of beta-amyloid (1–42) on poly-mer cushioned membranes: A combined study by neutron reflec-tometry and AFM force spectroscopy. Biochim Biophys Acta 808:1–10.

Ehrenhofer U, Rakowska A, Schneider SW, Schwab A, OberleithnerH. 1997. The atomic force microscope detects ATP-sensitive proteinclusters in the plasma membrane of transformed MDCK cells. CellBiol Int 21:737–746.

Fotiadis D. 2012. Atomic force microscopy for the study of membraneproteins. Curr Opin Biotechnol 23:510–515.

Fotiadis D, Scheuring S, Muller SA, Engel A, Muller DJ. 2002. Imag-ing and manipulation of biological structures with the AFM.Micron 33:385–397.

Frankel DJ, Pfeiffere JR, Surviladze Z, Johnson AE, Oliver JM, Wil-son BS, Burns AR. 2006. Revealing the topography of cellularmembrane domains by combined atomic force microscopy/fluores-cence imaging. Biophys J 90:2404–2413.

Frederix PL, Bosshart PD, Engel A. 2009. Atomic force microscopy ofbiological membranes. Biophys J 96:329–338.

Galietta LJ, Moran O. 2004. Identification of CFTR activators andinhibitors: Chance or design? Curr Opin Pharmacol 4:497–503.

Garcia R, Herruzo, ET. 2012. The emergence of multifrequency forcemicroscopy. Nat Nanotechnol 7:217–226.

Kiselyova OI, Yaminsky IV, Ivanov YD, Kanaeva IP, Kuznetsov VY,Archakov AI. 1999. AFM study of membrane proteins, cytochromeP450 2B4, and NADPH–Cytochrome P450 reductase and theircomplex formation. Arch Biochem Biophys 271:1–7.

Kraus B, Ziegler M, Wolff H. 2007. Linear fluorescence unmixing incell biological research. Mod Res Educl Top Microsc 1:863–872.

Le Grimellec C, Lesniewska E, Giocondi M-C, Finot E, Vi�e V, Goudon-net J-P. 1998. Imaging of the surface of living cells by low-force con-tact-mode atomic force microscopy. Biophys J 75:695–703.

Mendes F, Farinha CM, Roxo-Rosa M, Fanen P, Edelman A, DormerR, McPherson M, Davidson H, Puchelle E, De Jonge H, Heda GD,Gentzsch M, Lukacs G, Penque D, Amaral MD. 2004. Antibodiesfor CFTR studies. J Cyst Fibros, 3:69–72.

Moller C, Fotiadis D, Suda K, Engel A, Kessler M, Muller DJ. 2003.Determining molecular forces that stabilize human aquaporin-1. JStruct Biol 142:369–378.

Monaldi I, Vassalli M, Bachi A, Giovedi S, Millo E, Valtorta F, RaiteriR, Benfenati F, Fassio A. 2010. The highly conserved synapsin do-main E mediates synapsin dimerization and phospholipid vesicleclustering. Biochem J 426:55–64.

Muller DJ. 1997. Adsorption of biological molecules to a solid supportfor scanning probe microscopy. J Struct Biol 119:172–188.

Muller DJ. 2008. AFM: A nanotool in membrane biology. Biochemis-try 47:7986–7989.

Fig. 10. Integrated optical-AFM setup. A: Fluorescence image of asample marked with the FM4-64 dye (yellow). B–D: Superposition ofthree AFM maps to the optical image. [Color figure can be viewed inthe online issue, which is available at wileyonlinelibrary.com.]



Orsini F, Santacroce M, Arosio P, Sacchi VF. 2010. Observing Xenopuslaevis oocyte plasma membrane by atomic force microscopy. Meth-ods 51:106–113.

Papi M, Maulucci G, De Spirito M, Missori M, Arcovito G, LancellottiS, Di Stasio E, De Cristofaro R, Arcovito A. 2010. Ristocetin-induced self-aggregation of von Willebrand factor. Eur Biophys J39:1597–1603.

Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, GrzelczakZ, Zielenski J, Lok S, Plavsic N, Chou JL. 1989. Identification ofthe cystic fibrosis gene: Cloning and characterization of comple-mentary DNA. Science 45:1066–1073.

Sbrana F, Bongini L, Cappugi G, Fanelli D, Guarino A, Pazzagli L,Scala A, Vassalli M, Zoppi C, Tiribilli B. 2007. Atomic force micros-copy images suggest aggregation mechanism in cerato-platanin.Eur Biophys J 36:727–732.

Schillers H. 2008. Imaging CFTR in its native environment. PflugersArch 456:163–177.

Schillers H, Danker T, Madeja M, Oberleithner H. 2001. Plasmamembrane protein clusters appear in CFTR-expressing Xenopuslaevis oocytes after cAMP stimulation. J Membr Biol 180:205–212.

Schillers H, Shahin V, Albermann L, Schafer C, Oberleithner H.2004. Imaging CFTR: A tail to tail dimer with a central pore. CellPhysiol Biochem 14:1–10.

Stahlberg H, Fotiadis D, Scheuring S, Remigy H, Braun T, MitsuokaK, Fujiyoshi Y, Engel A. 2001. Two-dimensional crystals: A power-ful approach to assess structure, function and dynamics of mem-brane proteins. FEBS Lett 504:166–172.

Wang H, Hao X, Shan Y, Jiang J, Cai M, Shang X. 2010. Preparationof cell membranes for high resolution imaging by AFM. Ultramicro-scopy 110:305–312.



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