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Force measurements on natural membrane nanovesicles reveal a composition-independent, high Young's modulus† a b c d Annalisa Calo ` ,* David Reguera, Gerard Oncins, Marie-Annick Persuy, d e e d Guenhae ¨l Sanz, Simona Lobasso, Angela Corcelli, Edith Pajot-Augy and Gabriel Gomilaaf

Mechanical properties of nano-sized vesicles made up of natural membranes are crucial to the development of stable, biocompatible nanocontainers with enhanced functional, recognition and sensing capabilities. Here we measure and compare the mechanical properties of plasma and inner membrane nanovesicles
80 nm in diameter obtained from disrupted yeast Saccharomyces cerevisiae cells. We provide evidence of a highly deformable behaviour for these vesicles, able to support repeated wall-to-wall compressions without irreversible deformations, accompanied by a noticeably high Young's modulus (
300 MPa) compared to that obtained for reconstituted artificial liposomes of similar size and approaching that of some virus particles. Surprisingly enough, the results are approximately similar for Received 24th September 2013 Accepted 2nd December 2013

plasma and inner membrane nanovesicles, in spite of their different lipid compositions, especially on

DOI: 10.1039/c3nr05107b

what concerns the ergosterol content. These results point towards an important structural role of membrane proteins in the mechanical response of natural membrane vesicles and open the perspective

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to their potential use as robust nanocontainers for bioapplications.

Introduction Nano-sized natural membrane vesicles constitute native nanocontainers of considerable fundamental and applied interest.

a

IBEC Institute for Bioengineering of Catalonia, C/Baldiri Reixac 10-12, 08028, Barcelona, Spain. E-mail: [email protected]; [email protected]; Fax: +34 934020183; Tel: +34 934031184

b UB University of Barcelona, Department of Fundamental Physics, C/Mart´ı i Franqu`es 1, 08028, Barcelona, Spain. E-mail: [email protected]; Fax: +34 934021149; Tel: +34 934039214 c

CCiTUB, Nanometric Techniques Unit, Scientic-technical and Technological Centers of University of Barcelona Services, C/Mart´ı i Franqu`es 1, 08028, Barcelona, Spain. E-mail: [email protected]; Fax: +34 934021398; Tel: +34 934020593

d INRA, UR1197 Neurobiologie de l'Olfaction et Mod´elisation en Imagerie, F-78350, Jouy-en-Josas, France. E-mail: [email protected]; guenhael.sanz@ jouy.inra.fr; [email protected]; Fax: +33 0134652241; Tel: +33 0134652564

Universit` a degli Studi di Bari Aldo Moro – Dipartimento di Scienze Mediche di Base, Neuroscienze ed Organi di Senso, Piazza G. Cesare, I, 70126, Bari, Italy. E-mail: [email protected]; [email protected]; Fax: +39 08054786109; Tel: +39 08054786108

e

UB University of Barcelona, Department of Electronics, C/Mart´ı i Franqu`es 1, 08028, Barcelona, Spain. E-mail: [email protected]; Fax: +34 934021148; Tel: +34 934020206

f

† Electronic supplementary information (ESI) available: Morphological nanovesicle data from AFM images and FS experiments, immunoblot analysis of sucrose density gradient membrane subfractions, thin layer chromatography (TLC) lipid analysis of membrane subfractions, derivation of the electrostatic contribution to force curves, verication of the thin shell model by nite element simulation. See DOI: 10.1039/c3nr05107b

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In biomedicine and in membrane protein-based biosensing, the isolation or the production of natural vesicles directly from a cell source is considered a strategy to massively obtain optimal membrane-based envelopes with incorporated biological functions. Even though this strategy is still in its infancy,1 natural nanovesicles possess a distinct advantage over synthetic liposomes as they contain all the membrane-associated proteins and lipids expressed by that particular cell line in their native conformation. The native lipid envelope provides a properly organized and uid environment where membrane proteins can undergo physiological conformational changes or interactions with ligand molecules maintaining their functional integrity.2 Working with natural vesicles also decreases the risk of protein denaturation as it is not necessary to reconstitute previously puried membrane proteins of interest into an articial liposome. Additionally, genetic engineering on the cell source allows the expression of cytoplasmic proteins or membrane receptors in nanovesicles;1 in this way, their surface and inner properties can be tailored for any number of desired applications, ranging from chemical sensing3,4 to delivery of therapeutics.5,6 From a fundamental point of view the study of nanovesicles derived from various cell compartments and membranes by spontaneous release to the extracellular medium7 or extruded from their plasma membrane8 or prepared as nanometric liposomes from the membrane fraction with no cytoplasmic content9,10 has become of increasing interest in the last few

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years. These nanovesicles, in fact, incorporate selected proteins, lipids and even RNA, which may consequently be released from the cell into the extracellular space. Their content depends on the subcellular origin and on the specic cell type and may reect the physical or the physiological/pathological status of the membrane they come from.11,12 Due to their small size, so nature and need to be maintained under an appropriate ionic solution, the characterization of the physical and chemical properties of nano-sized membrane vesicles constitutes a real challenge. Among various physical properties of interest, the mechanical properties of nano-sized membrane vesicles are considered to be of outmost importance, as they crucially affect the stability and functionality of the vesicles. For example, in the eld of biomedicine the tissue permeability and the efficiency to intercalate and release biomolecules with a retained functionality are expected to be enhanced for elastic, highly deformable vesicles whose membrane is in a uid state,13 as it has been observed for synthetic liposomes.14–16 Ultra-deformable sub-100 nm vesicles are believed to combine the structural stability due to their size with optimal adsorption and integration with living tissues.17,18 Furthermore, nano-sized vesicles keeping the same cytoplasmic content as the whole cell can be considered as a miniaturized version of the cell whose mechanical properties are expected to be similar to those shown by highly curved regions of cells appearing in processes like endo- and exocytosis, adsorption on irregular surfaces and cell motility.19–21 Thus, they can be used as model systems to assess, for example, the effect of the membrane composition and its lateral organization on the vesicle bending rigidity, or to extract parameters to be processed in simulations to study the interplay between the regulation of mechanical tension in cells and the mechanical properties of highly curved invaginations spontaneously formed at its surface.22 Finally, the mechanical properties are also of interest in practical applications of these nanoscale vesicles for biosensing since they can provide information on their deformation properties and stability upon adsorption onto biosensor surfaces, thus integrating morphological and functional characterizations.10 At present, reports on the mechanical properties of nanovesicle membrane structures have been presented only for the case of synthetic23–25 and semi-synthetic26 lipid envelopes in a much simpler composition and phase state. Recently, Schaap and co-workers studied the mechanical behaviour of the lipid envelope obtained by inuenza virions budded from the plasma membrane of infected cells.27 Similarly, the mechanical properties of viral particles and capsids in their natural conformation have also been investigated.28,29 These studies have shown remarkable differences between the Young's modulus of liposomes and viral particles, in the range 10–100 MPa for the former and 300 MPa–3 GPa for the latter, revealing an important role of composition and structure in the mechanical properties of these nanoscale objects. Whether the composition and structure are also relevant to natural cell membranes, which contain a signicant proportion of both lipids and proteins, is still unknown.

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In this work we investigate, precisely, the mechanical properties of natural nano-sized vesicles (diameter
80 nm), obtained from two different membrane fractions of disrupted yeast Saccharomyces cerevisiae cells that carry heterologously expressed olfactory receptors30 and contain buffer in their interior. Two different membrane fractions (corresponding to sub-fractions of plasma and inner membranes) have been studied to elucidate the effect of the biochemical composition on the mechanical response, in particular on what concerns the ergosterol content. We chose this particular type of natural nanovesicle for its relevance in the development of odorant biosensors based on olfactory receptors.3,9 Mechanical properties are investigated by means of force spectroscopy measurements with an atomic force microscope (AFM). We show that natural vesicles from Saccharomyces cerevisiae, in spite of their intrinsic compositional complexity and structural organization, can be characterized by force spectroscopy similarly to reconstituted phospholipid liposomes and virus particles. In particular, our results show that such nanovesicles are highly deformable, i.e. they can be deformed across their whole thickness, recovering completely their shape aer multiple wallto-wall indentations by the AFM probe. The mechanical response can be quantitatively interpreted with the support of nite element numerical simulations, in order to extract reliable values of the Young's modulus. Importantly, we show that the Young's modulus takes a remarkably high value around 300 MPa, a fact that suggests a relevant role of membrane proteins, in comparison to lipids, in determining the mechanical response of natural cell membranes. This fact is further supported by the independency of the extracted Young's modulus from the lipid composition of the membrane vesicles studied (plasma and inner membranes).

Results General properties of force curves on natural nanovesicles In the present study, diluted solutions of nanovesicles (NV) obtained from plasma and inner Saccharomyces cerevisiae membrane fractions were deposited onto hydrophilic glass, as detailed in the Experimental section. Once deposited, nanovesicles partially atten on the surface and they can be recognized by intermittent contact mode AFM as rounded nanoobjects in topographic images (Fig. 1a). Nanovesicles from two different membrane sub-fractions have been analyzed, namely, membranes from sub-fraction 13 and sub-fraction 2, according to their fractionation in a sucrose gradient. The former subcellular fraction corresponds to the plasma cell membrane while the latter to the inner cell membrane (presumably from vacuoles), as it has been previously reported31 (see also ESI† and the Experimental section). We analyzed a set of force curves collected on a number of different nanovesicles from the plasma membrane fraction and from the inner membrane fraction. The analyzed nano-vesicles showed height and width values in the range hNV ¼ 14–55 nm and wNV ¼ 64–294 nm respectively, as determined from topographic images. Cryo-EM inspection of nanovesicles from crude yeast membranes before subcellular fractionation showed

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Force curves on a nanovesicle from the inner yeast membrane fraction. All curves show a linear behaviour for small indentations (d # 20 nm). Curves of type 1 show a change of slope at high indentations and do not show any breakthrough event. Curves of type 2 show instead two breakthrough events. Curves of type 3 only show one breakthrough event at high d values while curves of type 4 show it immediately before the contact with the hard substrate. (Inset) Schematic representation of the force spectroscopy experiment showing the AFM tip on the nanovesicle surface at two levels of magnification, where the relative sizes of both interfaces is accounted for.

Fig. 2

Tapping-mode AFM topography of a nanovesicle from the plasma membrane fraction at the beginning of the force spectroscopy (FS) experiment (a), after 17 force curves (b) and after 58 force curves (c). (d) Topographic profiles corresponding to the lines in the middle of the vesicle. Topographically measured hNV values and wNV values were 29 and 88 nm (a), 33 and 86 nm (b), 31 and 91 nm (c) respectively. The Z scale for all the images is 35 nm. Fig. 1

spherical particles with an average size between 50 and 200 nm.10 Topographic data for each vesicle are reported in Table S1 of the ESI.† The mechanical properties of single nanovesicles have been obtained from the AFM force spectroscopy (FS) curves,32,33 as detailed in the Experimental section. In a typical approach force curve, the tip is brought into mechanical contact with the sample and proceeds until it reaches the hard substrate. The resulting membrane elastic deformation is followed by the AFM probe deection. In the case of synthetic liposomes the mechanical ngerprints usually extracted are the Young's modulus or the bending rigidity, normally obtained for small vesicle deformations,25 and the breakthrough forces, corresponding to the minimum force required to puncture the vesicle membrane.26 A schematic drawing of a FS experiment is reported in the inset of Fig. 2. In order to ensure that no plastic deformation or local damage was provoked on the vesicles during FS experiments,34 intermittent contact mode AFM images of individual nanovesicles were acquired before and aer each set of nanomechanical tests. The obtained results conrm that nanovesicles recover their initial hNV and wNV aer multiple wall-to-wall compressions (hard contact between the tip and the substrate was conrmed by the presence of a vertical region in the force vs. indentation, F vs. d, curve)35 and that their morphology does not dramatically change. Fig. 1 shows, as a typical example, an AFM image of an individual nanovesicle from the plasma membrane fraction at the beginning of the

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experiment (Fig. 1a), aer 17 (Fig. 1b) and aer 58 (Fig. 1c) force curves at a maximum vertical force of 5 nN collected on top of it. Aer the rst series of force curves, the nanovesicle shape and its heterogeneous features remain essentially unaltered (see Fig. 1b), while structural changes are visible aer 58 puncturings (see Fig. 1c). Nevertheless, the vesicle-like structure is still conserved and it does not break. Based on these results, and in order to prevent the occurrence of irreversible damage on the nanovesicles, the size and shape of the vesicles have always been monitored by AFM topography to not show major irreversible modications. Typical F vs. d curves collected on top of a single nanovesicle from the inner membrane fraction are shown in Fig. 2. They show an inherent remarkable variability, especially in the region of large indentations, if compared with curves collected on synthetic liposomes.23–25 The curves show an initial linear F vs. d evolution for d values #20 nm. Aer this initial linear regime, most of the curves (37.5% of the recorded curves) show a change of regime, i.e. a change of slope at a nanovesicle deformation of
45% that extends for 10–20 nm and ends up when the tip contacts the substrate. This is the case of curve 1 shown in Fig. 2. Variations of this type of curve include sudden, oen negative,26 changes of slope that extend for a few nanometers, located approximately at the intersection between the two regimes aer the initial linear part (8.5% of the cases, see curve 3 of Fig. 2) or immediately before the tip contacts the hard substrate and the F vs. d curve becomes vertical (11% of the cases). We interpret these jumps as breakthrough events of the nanovesicle membrane. In the case of supported planar lipid bilayers (SPBs), they have been previously used to quantitatively measure the maximum force that the membrane is able to withstand before being indented by the cantilever tip, magnitude that has been used as a gure of merit to report the Nanoscale, 2014, 6, 2275–2285 | 2277

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mechanical stability of the bilayer.36 Curves showing two discrete penetration events, corresponding to the breakthrough of the nanovesicle upper membrane and the membrane in contact with the substrate, like curve 2 of Fig. 2, represent 12% of the collected curves. The remaining 31% of the collected curves shows a rather monotonic F vs. d prole that possibly includes one breakthrough event at the intersection between the linear and the vertical part of the curve (see curve 4 of Fig. 2). Representative curves shown in Fig. 2 are reproduced similarly for all nanovesicles analyzed in the present study. The results reported in this paper derive only from the analysis of force curves like the ones shown in Fig. 2. Aberrant curves, i.e. which do not exhibit breakthrough events and/or a F vs. d evolution along the whole nanovesicle depth, were disregarded and not used for further analysis.

Geometric and structural properties obtained from force curves on single natural nanovesicles The Young's modulus evaluated on natural membranes sensibly depends on the internal structure of the nanovesicle (e.g. thickness of the membrane, the presence of an internal pressure) as well as on its actual geometrical dimensions. Both structural and mechanical properties of the nanovesicles can be obtained from the analysis of force curves. For instance the total indentation, measured from the beginning of the mechanical contact between the tip and the nanovesicle until contact with the underlying hard substrate,35 gives the height of the nanovesicle, while the vertical distance associated with breakthrough events should match roughly the thickness of the nanovesicle membrane. Thus, an analysis of the complete force curve evolution allows the extraction of geometrical parameters of nanovesicles, such as their height hNV and the thickness of their membrane envelope t. As we will show, for very so nanoscale objects like membrane nanovesicles, height evaluation from force curves provides more accurate values than that obtained from topographic images. In Fig. 3a the vertical distance from the beginning of the cantilever deection until the contact with the glass surface (hNV (FS)) is reported vs. the corresponding nanovesicle height obtained from the topographical prole in the middle of each nanovesicle at the beginning of the FS experiment (hNV (topography)). We found that hNV (FS) values exhibit a certain variability during the ongoing FS experiment on each individual nanovesicle and that the average value is statistically 24% higher than hNV (topography), as can be seen in the linear t reported in the gure. An underestimation of hNV by intermittent contact mode AFM topography is indeed possible due to the sample soness. This fact renders critical the choice of the optimal amplitude setpoint for vesicle imaging,37 which is related to the vertical force applied on the nanovesicles by the AFM probe. As an example, the height deviations up to 50% have been reported for biological molecules when applying variations of the amplitude set-point in a reasonable range38 and, depending on the local mechanical properties, in polymer samples apparently smaller heights have been observed in so

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regions compared to harder regions of the same sample.39 By xing in 1 the slope of the linear t in Fig. 3a the obtained intercept A ¼ (6  2) nm can be considered an estimation of the actual deformation exerted on the nanovesicles by the AFM tip during scanning. This deformation would correspond to an applied force on the order of 0.25 nN.40 The choice of the contact point can also be critical when analyzing force curves because short range electrostatic repulsions, hydration or steric forces41 can provoke tip deection before the mechanical contact with the vesicles. According to this, hNV (FS) can be an overestimation of the real value. We veried that in this particular case the force produced from overlapping the electrical double layers of the tip apex of the AFM probe and of the nanovesicle surface induces a negligible cantilever deection (see Fig. S2 of the ESI†). Thus it can be assumed that under the present experimental conditions the force curve evolution is entirely due to the nanovesicle mechanical deformation. From these considerations, we used the hNV (FS) values from the force spectroscopy experiment and not the topographical value for the extraction of the Young's modulus of the vesicles. In Table S1 of the ESI† the average hNV (FS) obtained for each nanovesicle studied is reported. Fig. 3c shows the histogram of the vertical distance associated with all the abrupt changes of the slope in the force curves (breakthrough events). These jumps measure ca. 5 nm, irrespective of their position along the force curve, i.e. at the transition between the two contact mechanics regimes or immediately before the contact with the hard substrate, and of the number of penetration events (see curves 2, 3 and 4 reported in Fig. 2). The average values obtained for nanovesicles from both membrane fractions are not signicantly different, being (5.0  2.3) nm and (4.9  2.0) nm for nanovesicles from the plasma and inner membrane fractions, respectively. These values correlate well with the thickness of the nanovesicle membrane (t) (
6 nm) obtained from topographic images of occasional membrane patches found in the sample (see Fig. 3d and inset).42 Mechanical properties obtained from force curves on single natural nanovesicles In order to extract the nanomechanical properties of the vesicles (e.g. Young's and bending moduli, E and kbend, respectively) force curves need to be analyzed in the framework of an appropriate theoretical mechanical model. We have modelled the nanovesicles as elastic thick shells, placed on a crushproof substrate and subject to large deformations by a spherical AFM tip. The nanovesicles absorbed on the substrate have been considered as spherical caps with a radius of curvature, RNV, being given by:43 RNV ¼

hNV 2 þ wNV 2 =4 2hNV

(1)

In Table S1 of the ESI† we report the radius of curvature of the nanovesicles analyzed in this study, whose value span a range 38–283 nm. For Young's modulus extraction we used the height obtained from force spectroscopy curves, hNV (FS), and

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(a) Nanovesicle height as measured by force spectroscopy vs. topographic height measured at the beginning of the experiment. The linear fit in black includes the experimental data for both membrane fractions. (b) F/E vs. d curve obtained by finite element simulations for a nanovesicle adsorbed as a spherical cap on a hard substrate and indented by a spherical tip of radius ¼ 9 nm (nanovesicle geometry: hNV ¼ 30 nm, wNV ¼ 120 nm, t ¼ 5 nm). The dashed lines represent the elastic and buckling regimes, respectively, with a crossover at indentations around the membrane thickness. Two cross-sections of the deformed nanovesicle are shown, together with the distribution of von Misses stress (in a color scale) corresponding to the two different regimes: small deformations, where the shape is conserved, and inverse buckling at indentations larger than the membrane thickness. (c) Histogram of the vertical distances associated with abrupt jumps (breakthrough events) occurring in the force curves. Data are presented separately for nanovesicles from the two analyzed membrane fractions. (d) AFM image showing an occasional membrane patch found in a sample of nanovesicles from the total membrane fraction. Inset: topographic profile corresponding to the white line in the middle of the membrane patch. Fig. 3

the width from topographic images, wNV, for the reasons discussed before. Using this geometry, we carried out nite element simulations of the F vs. d experiments in order to analyse the nanovesicle mechanical response and its dependency on the different geometrical and mechanical parameters (see details of the simulation in the Experimental section). All the calculated curves scale with the Young's modulus, E, and they merge into a single master curve when F/E is plotted vs. d. In Fig. 3b the master curve obtained with parameters corresponding to the average nanovesicle dimensions, i.e., hNV ¼ 30 nm, wNV ¼ 120 nm, t ¼ 5 nm and tip radius Rtip ¼ 9 nm is shown, together with an image showing the shape evolution of a section of the nanovesicle during indentation and the local stress on its wall. The curve predicts an initial linear deformation regime followed by a second regime where F scales as d1/2 at higher d This journal is © The Royal Society of Chemistry 2014

values, corresponding to approximately one-two times the membrane thickness. This change of regime is associated with a mirror buckling of the top part of the nanovesicle (see the nanovesicle sections in Fig. 3b).44,45 Buckling at large indentations has been found in all our calculations, which have been performed with a wide range of tip sizes and shapes. We veried that the slope of the initial region of linear deformation is almost insensitive to the tip radius, increasing by less than 20% when the tip radius is increased from 4 to 50 nm.34 We also repeated the simulations for different shell thicknesses and geometries (see Fig. S3 and S4 of the ESI†). As the thickness of the shell is increased, the force curves at small indentations become progressively more non-linear, approaching the limiting F vs. d3/2 scaling expected according to the Hertz model of a tip indenting a quasi-spherical homogeneous material.46 Coherently to previous ndings,26 the Nanoscale, 2014, 6, 2275–2285 | 2279

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Hertzian regime spans a range of indentations that depends on the tip geometry, being more noticeable for indentations with cylindrical tips, as well as for indentations between the at plates. It can be shown (see also the ESI†) that the results obtained from the nite element simulations of nanovesicles with shells of different thicknesses and radius of curvature in the range of those of the natural membrane nanovesicles in the present study and for small indentations (d # t) can be described with good accuracy by the thin shell formula:47 F ðdÞ ¼ kNV d with

kNV ¼

aEt2 RNV

(2)

a being a geometry-dependent proportionality factor that includes the Poisson ratio n, which we take as n ¼ 0.5.34 Estimates of the slope of the linear region in Fig. 3b give a ¼ 1.73.34 Similar predictions have been shown to apply also to ellipsoidal shells,48,49 with a different proportionality factor a. The agreement with the thin shell predictions is remarkable, since the shell is not strictly thin (in the sense of t/RNV  1) and the deformations explored are not small, and it has also been observed in the case of indentation of viral capsids.47,50 Thus, the Young's modulus E can be extracted from the slope of the initial linear region of the experimental curves according to eqn (2), using the average thickness t from force curves (see Fig. 3c) and the radius of curvature RNV from eqn (1), with the height hNV (FS) values from force curves (see Fig. 3a) and taking wNV from the topographic prole in the middle of the nanovesicles at the beginning of the experiment. Fig. 4 shows representative force curves together with the linear ts for the small deformation region (continuous lines). In the gure we also identify the region of large deformations as dot-dashed lines with a reduced slope (see the Discussion section). In Fig. 5 the E values obtained for each nanovesicle analyzed are reported. The values obtained are relatively homogeneous in spite of the variability in the nanovesicle geometry and in the recorded force curves (see Fig. 2 and 4) and apparently they are not correlated with the specic geometry of the nanovesicles. These facts support the idea that the obtained Young's modulus represents an intrinsic mechanical property of these natural membranes. The average values for the two populations of nanovesicles (from plasma and inner membrane fractions) give E ¼ (336  229) MPa and E ¼ (237  99) MPa respectively, with a not signicant dependency on the composition of the two membranes. For comparison, in Fig. 5 we also plot the Young's modulus reported in the literature for some liposomes and viral particles. The corresponding bending rigidity:26 kbend ¼

Et3 24ð1  n2 Þ

(3)

is, respectively, (2.3  3.6)  1018 J (z560 kT) and (1.5  2.0)  1018 J (z370 kT). The extracted E value is predicted to be sensitive to the membrane thickness t, i.e. being
30% lower when the membrane thickness is increased 1 nm, that is, from 5 nm to 6 nm, and it depends on the assumption made about the

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Typical F vs. d curves and corresponding linear fits in the small deformation regime (continuous line in grey) according to the thin shell model described in the text. The region of the change of regime is indicated as a dot-dashed line in grey, and the breakthrough events are indicated with grey arrows. Curves on the left refer to nanovesicles from the plasma membrane fraction, while curves on the right refer to nanovesicles from the inner membrane fraction.

Fig. 4

nanovesicle geometry. Indeed, if we consider an ellipsoidal geometry for the nanovesicles,48 at least for the boundary case hNV  wNV, the curvature radius at the indentation point for the same topographical features is expected to be higher by a factor
4 compared to a spherical cap geometry and gives E ¼ (1.94  1.57) GPa and E ¼ (1.32  0.60) GPa, respectively, for nanovesicles from the plasma and the inner membrane fractions. These values are unreasonably high compared to the reported data for lipid vesicles25,26 and could support the assumption that the nanovesicles are adsorbed as nearly spherical caps on the substrate rather than just being slightly deformed on it. Indeed, the spherical cap conformation is predicted to set up in the case of strong adhesion of uid membranes and vesicles on a at substrate.51 Finally, we analyzed the characteristic force at which nanovesicles show a change of mechanical regime (FC), i.e. the force at which the indentation curve deviates from the initial

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Fig. 5 Calculated Young's modulus, E, for each nanovesicle (solid red and blue symbols). Data in red refer to nanovesicles from the plasma membrane fraction, while data in blue to nanovesicles from the inner membrane fraction. For comparison, the Young's modulus of some virus particles and liposomes, taken respectively from ref. 26 and 28, is also reported. The error bars correspond to one standard deviation obtained by analyzing between 10 and 20 curves per nanovesicle.

linearity, as well as the breakthrough forces at which sudden penetration events are displayed in the force curves (Fy). We refer to Fy1 as the force corresponding to the puncturing of the upper nanovesicle membrane and to Fy2 as the force corresponding to the second membrane rupture before the tip gets in contact with the substrate. Fig. 6a shows, separately for each membrane fraction, the histogram of FC in curves that do not show any breakthrough event at the change of regime (48.5% of the collected curves). The values we obtain, on the order of 1.5–1.8 nN for both types of nanovesicles analysed, are not signicantly different and coincide with the average force required to puncture the upper nanovesicle membrane (see Fig. 6b and c). Fy2 is statistically about 0.5–1 nN higher than FC, a fact that could reect the role of the substrate in increasing the breakthrough force. The values of breakthrough forces here reported are in the order of those obtained on lipid vesicles from inuenza virus.26

Discussion We found that the thin shell model, which has already been used to predict the mechanical behaviour of indented viral

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capsids34,47 and envelopes26,27 as well as that of polymer vesicles43,52 of size around 100 nm, reproduces the evolution of the experimental force curve for small indentations (d # t) and allows extraction of a reasonably precise value of the Young's modulus beyond the force curve variability and geometrical differences among nanovesicles. The rst interesting conclusion obtained from the force curve analysis is that the mechanical properties of the plasma and inner membrane nanovesicles do not show a signicant difference within the dispersion of the measurements. This is especially relevant due to expected differences of the two types of membranes from the structural and chemical point of view.53 Immunoblot analysis of the membrane fractions and Thin Layer Chromatography (TLC) of their lipid extracts show in fact that only nanovesicles from the plasma membrane contain lipid ras and that in these vesicles the ergosterol content is higher compared to polar lipids (see Fig. S1b of the ESI†), thus conrming the literature data.31,54 The ergosterol content in the yeast plasma membrane fraction (expressed as molergosterol/ molphospholipid), in particular, has been reported to be 18 times higher compared to its amount in vacuoles.55 Ergosterol is expected to play a role similar to cholesterol in higher eukaryotic cells, whose controlled addition in EggPC56 and DMPC26 liposomes has been proven to increase the membrane rigidity in force spectroscopy experiments. Indeed, AFM topography and force measurements show that the expected differences in the two types of nanovesicles, concerning the chemical composition or the possible presence/absence of stiffer gel domains,53 neither results in a different nanovesicle size (equivalent sphere diameter) or shape (aspect ratio, radius of curvature), once they are deposited on the glass surface. These geometrical parameters are expected to play a major role in determining the force curve evolution according to the thin shell model. The Young's modulus we measured, 200–300 MPa, is more than four times higher than the average value obtained on reconstituted lipid vesicle systems (see Fig. 5). By modelling the vesicles of size around 100 nm made up of the lipid envelope of inuenza virus as spherical shell resting on a rigid at surface, Li et al.26 extracted an E value of 45 MPa at room temperature while, applying the thin shell model, Delorme et al.25 obtained an E value of 110  10 MPa for reconstituted unilamellar DPPC liposomes with a spherical cap

Fig. 6 (a) Histogram of the force at which the change of regime is observed (FC). Extracted FC (open circles), Fy1 (solid circles) and Fy2 (solid triangles) for each nanovesicle from the plasma membrane fraction (b) and from the inner membrane fraction (c).

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shape. The high E value obtained in this case can be explained as the DPPC phospholipid membrane is in a solid (gel) phase at room temperature.26 These values are well above the corresponding values of
1 MPa obtained for synaptic vesicles46 and eggPC vesicles23 of the same size analyzed according to the Hertz model that underestimates E as it does not account for the unique hollow shell structure of these vesicles.43 If analyzed as thin shell structures, the Young's modulus in these cases would increase roughly by a factor of ten, still being much smaller than the values we found for the case of natural nanovesicles. It is remarkable that the extracted values for natural vesicles are somehow intermediate in between those found in synthetic liposomes and in viruses comprising a protein shell possibly lled with a densely packed genetic material (see Fig. 5). For viral particles, Young's moduli between a few GPa for ellipsoidal double-stranded DNA bacteriophage phi2950 and of 140 and 190 MPa for empty and full viral capsids of cowpea chlorotic mottle virus (CCMV), a single-stranded RNA virus modelled as a spherical shell,34 have been reported.28,29 Our results point towards an important structural role of the yeast membrane proteins in supporting the membrane vesicle envelope beyond their traditionally recognized role in transport, signal transduction or in specic cell metabolic functions.42 In principle, this fact should not be surprising in view of their relatively high content in natural membranes (expressed in mgprotein/mgphospholipid) which oscillates between
4 (plasma membrane fraction) and
2 (vacuoles).55 The high stiffness of the natural vesicles is a characteristic that can be compatible with a still uid nature of their membrane. Concerning the phase of the nanovesicles envelope, the fact that they remain fully and reversibly deformable aer many wall-to-wall indentations suggests that their envelope is rather uid at room temperature. Indeed, measurements of membrane uidity in whole cells of Saccharomyces cerevisiae report a phase transition temperature of 12  C in aqueous solution at an osmotic pressure of 1.38 MPa.57 The total lipid extracts from the plasma membrane from Saccharomyces cerevisiae exhibit a phase transition at 20  C in pure water.58 A nal remark concerns the behaviour of the force versus indentation curves at large indentations (d
20 nm), where a second nearly linear regime seems to be shown (see the dashed lines in Fig. 4). The slope of this second linear region of the force curves is statistically 35% lower compared to the slope of the initial part. What causes this behaviour is not clear at present. From a qualitative point of view one could argue that buckling phenomena take place, which would in turn give rise to a milder F vs. d dependence as F f d1/2 and account for the observed roughly linear behaviour. However, this interpretation does not quantitatively agree with the numerical simulations, which show the onset of buckling effects for indentations of the order of the shell thickness (
5 nm) (see Fig. 3b), while the experimental curves show a change of regime at indentations sensibly larger (15–20 nm). Possible reasons why the nanovesicles seem to show an enhanced resistance to buckling could include some internal reorganization during indentation or the presence of tension induced by their strong adsorption on the

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substrate. Pressurization, potentially coming from an osmotic origin, is also known to increase the rigidity and delay the onset of buckling in elastic shells.49 By numerical simulations we estimate that a very large osmotic pressure on the order of tens of atmospheres would be required to justify the observed behavior. Further research is necessary to clarify this point. Noticeably, our results show that the force at which the force curves show a change of the slope (without ruptures, see curve 1 in Fig. 2), FC, practically coincides with the minimal force necessary to generate sudden penetration events in the force curves (Fy1). This result could support the assumption that at the change of the mechanical regime the membrane is probably punctured, even if a clear breakthrough event is not always observed. A possible tip entrapment into the vesicle lipid envelope in this case could be envisioned as the onset of the change of regime, alternatively or simultaneously with buckling.

Experimental Subcellular fractionation of yeast membranes by sucrose density gradient and nanovesicle (NV) solution preparation Crude membranes from 50 mL of induced yeast were prepared by mechanical disruption of yeast cells in a lysis buffer, and then centrifugation, which allows recovering a fraction enriched in cellular membranes, as previously described.30 The membrane pellet was suspended in 500 mL of 10% sucrose, 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor cocktail (Complete, Roche) (108 h of induction in galactose medium at 15  C). 500 mL of membranes were layered on top of a continuous gradient of 30–70% sucrose in 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor cocktail. Gradients were centrifuged for 16 h at 30 000 rpm in a Beckman SW 41Ti rotor at 4  C. 13 fractions were collected from top to bottom of the gradient and kept at 80  C.59 Immediately before use, the stock suspension of membrane fractions 13 and 231 was diluted to a concentration (expressed as total protein concentration TPC) of 40 mg mL1 in phosphate buffered saline (PBS 1) 10 mM (pH ¼ 7.4) (Sigma Aldrich) and sonicated for 20 min at 50 kHz (Ultrasons, Selecta) in ice-cold water to homogenize the vesicle size. The solution was diluted in PBS to TPC ¼ 10 mg mL1 and then ltered using a sterile low binding protein lter (diameter ¼ 13 mm; pore size ¼ 0.22 mm) (Millipore). The NV solutions described in the text were obtained by further dilution with PBS to a nal concentration of 5 mg mL1. Immunoblot analysis of subcellular fractions Fractions of sucrose density gradients were analysed by immunoblotting. Electrophoresis was performed on 10% SDS polyacrylamide gels, electrotransferred onto ImmobilonTM Transfer Membranes (Millipore) and hybridized with antibodies targeting the cmyc epitope tagging ORs (anti-cmyc 1 : 1000, Roche), the yeast lipid ras and plasma membrane marker Pma1 (anti-Pma1 1 : 50 000, Abcam), the yeast endoplasmic reticulum membrane marker Dpm1p (anti-Dpm1p 4 mg

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mL1, Molecular Probes) and the anti-Vps10p yeast Golgi marker (4 mg mL1, Molecular Probes). Secondary antibodies used were peroxidase-conjugated anti-mouse (1 : 2000) or antirabbit (1 : 5000) IgG antibodies (Sigma). An enhanced chemiluminescence detection system (Perkin Elmer) and BioMax Light Films (Kodak) were used to reveal targeted proteins. Thin-layer chromatography (TLC) analyses of lipids of membrane fractions Total lipids were extracted using the Bligh and Dyer method, as modied for extreme halophiles.60 All organic solvents used throughout the study were commercially distilled and the highest available purity (Sigma-Aldrich). The extracts were dried under N2 before weighing and then dissolved in chloroform. Total lipid extracts were analyzed by TLC on silica gel 60A plates (Merck, 20  10 cm2, layer thickness 0.2 mm). The plates were washed twice with chloroform–methanol (1 : 1, vol/vol) and activated at 180  C before use. Cellular lipids were separated by preparative TLC on Silica gel 60A plates (20  20 cm2, layer thickness 0.5 mm) eluted with Solvent A (chloroform–methanol–acetic acid–water, 85 : 15 : 10 : 3.5, by vol.). Six main bands of polar lipids were observed. Aer scraping the silica in each band from the plate, the lipids were extracted ve times with chloroform–methanol (1 : 1, vol/vol), and the combined supernatants were brought to dryness under a stream of nitrogen.60 The six lipid bands were then re-chromatographed in Solvent B, of composition chloroform–methanol-29% ammonium hydroxide, 65 : 35 : 5, by vol. The following stains were used for the detection of lipids: (a) 5% sulfuric acid in water, followed by charring at 120  C;60 (b) iodine vapor;60 (c) molybdenum blue spray reagent (Sigma) specic for phospholipids. Sample preparation and AFM imaging Glass slides (diameter ¼ 25 mm, thickness ¼ 0.13–0.16 mm) (Menzel–Gl¨ aser) were sonicated in ethanol–water (50 : 50, vol/ vol) for 10 min and dried with pure nitrogen. They were then kept in an UV/ozone cleaner (Bioforce Nanosciences) for 10 min, in order to remove the organic contamination, before incubating the nanovesicle solutions. The glass RMS roughness, as measured by AFM, was (0.29  0.02) nm. 40 mL of a NV solution at a concentration of 5 mg mL1 were deposited on an area of 1  1 cm2 of cleaned glass slides for 15 minutes. This area was delimited by using a water repellent ink (Dako Pen). Aer incubation, the surface was rinsed 3 times with PBS and stored in PBS solution. For each membrane fraction we collected an AFM image of 10  10 mm2 (256  256 pixels) in order to individuate various NVs on glass and then a detail of size #1 mm2 (64  64 pixels) on individual NV before the force spectroscopy experiment at the scan rate of 3 lines per s. The cantilever was oscillated at 0.5 V (free amplitude) and the images were collected at the highest set-point voltage compatible with the images quality. Images were acquired before and aer FS experiments to ensure that there was no XY-piezo dri and to verify that the NV shape and size did not change due to the indentation by the AFM probe.

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The images were acquired with a MFP-3D AFM (Asylum Research, Santa Barbara, CA) in tapping mode in liquid, using triangular Si3N4 cantilevers with a nominal spring constant of 0.02 N m1 (OMCL-TR400PSA-1, Olympus, Mannheim, DE). In order to extract mean height and width of studied NV, higher resolution AFM images of the individual NV (256  256 pixels) were obtained and plane-tted using the WSxM soware (Nanotech, Madrid, ES).61 Assuming that NVs deform as spherical caps when deposited on glass from the solution, their volume was extracted from their height from force curves (hNV(FS)) and topographical width (wNV) as:10     1 wNV 2 hNV  V ¼ phNV 2 þ hNV 2 (4) 2hNV 4 3 By knowing the nanovesicle volume, the diameter of the equivalent sphere originating the spherical cap was extracted as 2(V3/(4p))1/3. The obtained average values, (70  26) nm and (83  29) nm for nanovesicles from the plasma and the inner membrane fractions respectively, are in agreement with reported nanoparticle tracking analysis (NTA) measurements that individuate a population of particles with size (94  20) nm in solutions of nanovesicles from the total membrane fraction of Saccharomyces cerevisiae.10 Force spectroscopy measurements FS experiments were performed on the central region of individual NV to avoid rim effects. A grid of force curves was drawn on top of the central region of chosen NV and force curves were performed on each position at a rate of 2 mm s1 and with a vertical maximum force of 5 nN (curve length: 200 nm). The individual spring constant of each AFM probe was calibrated by the thermal noise method implemented using the microscope soware (MFP3D 090909+1409, Asylum research, Santa Barbara, CA). By means of sensitivity calculation on a reference glass substrate, force curves were converted into F vs. d curves. Finite element simulations Finite element simulations have been performed using COMSOL Multiphysics v4.3 (Comsol, Stockholm, Sweden) to model the mechanical response of the NV. The NVs were modeled as thick shells with the shape of a spherical cap of external height hNV and width wNV adsorbed on top of a rigid at substrate. The membrane wall was considered as a homogeneous material with Young's modulus E and Poisson ratio n ¼ 0.5. The AFM tip was modeled as a sphere of radius Rtip ¼ 9 nm and very high Young's modulus. Taking prot of the axial symmetry along the z axis, only a section of the model was simulated as shown in Fig. 3b. The model was meshed with over 20 000 triangular elements. The contacts between the shell and the tip and the supporting surface during indentation were implemented with a contact-penalty stiffness method according to the manufacturer's manual. A parametric, non-linear solver was used to simulate the stepwise lowering of the tip onto the model. The nite element simulations performed in this work do not use the thin shell approximation. The 3D equations of elasticity are

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solved and the shell is discretized using tetrahedral elements. In the simulations, the boundary conditions are that the surface of the membrane in contact with the substrate cannot unbind (i.e. the vertical displacement of the surface of the membrane in contact with the substrate is xed to zero). Instead, the surface of the membrane in contact with the substrate is free to deform and stretch radially and the angle is not xed. The NV equivalent spring constant was obtained from a linear t of the force versus indentation curves, for small indentations.

Conclusions Force spectroscopy coupled with nite element calculations have been used in this work to investigate the mechanical properties of natural nano-sized vesicles produced from yeast cells expressing olfactory receptors at their surface, in addition to all the proteins which are naturally present in the yeast membrane envelope. We obtained a similar value of the effective Young's modulus for vesicles from different membrane fractions (plasmatic and inner yeast membranes) irrespective of the overall nanovesicle geometry (curvature at the indentation point, membrane thickness). The remarkably high E value experimentally measured for these vesicles, compared to other phospholipid liposome systems, and approaching the values obtained for some viruses of similar size, underlines the role of the membrane proteins in conferring mechanical stability to the nanovesicle lipid bilayer and may have implications in physiological processes involving nanovesicles. The present results validate the use of force spectroscopy for the nanoscale mechanical characterization of natural membrane vesicles.

Acknowledgements The authors are grateful for nancial support from the Spanish MINECo under Grants no. TEC2010-16844 and FIS2011-22603, and from the European Commission under Grant no. NMP228685-2, from Agence Nationale de la Recherche (NOSE, ANR07-PCVI-0027-01), and from the Project no. 1353, funded by the General Defence Secretariat/National Armaments Directorate, of the Italian Ministry of Defence, in the framework of the National Military Research Plan (PNRM). The authors thank Dr Xavier Sisquella (Parc Cient´ıc, UB) for fruitful discussions in AFM and FS measurements and Maristella Baronio for suggestions in TLC analyses.

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