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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Acta Biomater. 2013 Sep 21;10(4):1663–1670. doi: 10.1016/j.actbio.2013.09.007

A Mussel-Derived One-Component Adhesive Coacervate

Wei Wei 1, Yerpeng Tan 2, N Martinez Rodriguez 3, Jing Yu 4, Jacob N Israelachvili 1,4,*, J Herbert Waite 1,2,3,*
PMCID: PMC3960351  NIHMSID: NIHMS530068  PMID: 24060881

Abstract

Marine organisms process and deliver many of their underwater coatings and adhesives as complex fluids. In marine mussels, one such fluid, secreted during the formation of adhesive plaques, consists of a concentrated colloidal suspension of a mussel foot protein (mfp) known as Mfp-3S. Results of this study suggest that Mfp-3S becomes a complex fluid by a liquid-liquid phase separation from equilibrium solution at a pH and ionic strength reminiscent of conditions created by the mussel foot during plaque formation. The pH dependence of phase separation and its sensitivity indicate that inter/intra-molecular electrostatic interactions are partially responsible for driving the phase separation. Hydrophobic interactions between the nonpolar Mfp-3S proteins provide another important driving force for coacervation. As complex coacervation typically results from charge-charge interactions between polyanions and polycations, Mfp-3S is thus unique in being the only known protein that coacervates with itself. The Mfp-3S coacervate was shown to have an effective interfacial energy of ≤ 1 mJ/m2 which explains its tendency to spread over or engulf most surfaces. Of particular interest to biomedical applications is the extremely high adsorption capacity of coacervated Mfp-3S on hydroxyapatite.

Introduction

One of the most fascinating aspects about the underwater adhesion of marine organisms such as mussels and sandcastle worms is its reliance on metastable, water-insoluble fluids that resist being dispersed in the surrounding seawater. In mussels, these adhesive fluids consist of highly concentrated, intrinsically unstructured polyelectrolytes known as mussel foot proteins (mfps) that rapidly solidify upon equilibration with seawater. In sandcastle worm cement, given the presence of both polyanions (polyphosphoserine-rich protein) and polycations (lysine-rich proteins), fluid-fluid phase separation is modelled as a complex coacervation leading to a polyelectrolyte-depleted equilibrium phase and a denser, protein-rich coacervate phase [1, 2]. Complex coacervation results from the coulombic attraction and neutralization of oppositely charged polyelectrolytes coupled with the concomitant release of counter micro-ions [3] and confers unusual properties on the coacervate phase including relatively high diffusion coefficients of the solute and solvent molecules, high concentrations, relatively low viscosity, and low interfacial energy - all highly conducive to dispensing adhesives underwater [4–8]. Coacervates are used industrially in micro-encapsulation technology [9, 10], and are particularly important in food processing, as well as drug and gene delivery [11–15]. Hydrogel formation can also be mediated by coacervation [16].

Polyanions are not known to be involved in mussel adhesion, thus the basis for fluid-fluid phase separation by mussel foot proteins (Mfp) remains unknown. In this report, we show that Mfp-3S (Figure 1), a zwitterionic protein functioning as both adhesive primer and sealant in mussel adhesion [17], undergoes fluid-fluid phase separation at conditions identical to those imposed by the mussel’s foot during plaque formation. The outstanding interfacial adhesive and cohesive properties of Mfp-3S over a relatively wide pH range have been demonstrated previously using a surface forces apparatus (SFA) [17], and attributed to its abundant 3, 4-dihydroxyphenylalanine (Dopa) content and unique hydrophobic sequence. The strategy of achieving efficient phase separation and surface spreading by coacervation is very appealing in its simplicity, in part, because it is only rarely observed in single protein solutions: only tropoelastin is known to undergo a simple hydrophobically driven coacervation [18, 19]. Mfp-3S provides an interesting counterpoint for understanding the molecular requirements for single component coacervation. Based on how pH, ionic strength, and temperature affect Mfp-3S coacervation, we propose that the electrostatic and hydrophobic driving forces are uniquely balanced in the observed fluid-fluid phase separation of Mfp-3S. These forces will, of course, be subjected to much greater scrutiny as synthetic and recombinant mimics become more readily available. The relevance of coacervates to orthopaedic and dental materials was explored by investigating adsorption of Mfp-3S coacervates to hydroxyapatite (Ca10(PO4)6(OH)2, HAP) surfaces.

Figure 1.

Figure 1

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Mfp-3S from mussel plaque and its representative sequence. Red, blue and green indicate positively charged, negatively charged and aromatic residues, respectively.

Experimental Section

Mfp-3S purification

Mfp-3S was purified from the plaques of the California mussels, Mytilus californianus as described elsewhere [20]. About 1000 accumulated plaques were thawed and homogenized in a small volume (5 ml/200 plaques) of 5% acetic acid (v/v) containing 8 M urea on ice using a small hand-held tissue grinder (Kontes, Vineland, NJ). The homogenate was centrifuged for 30 min at 20,000 × g and 4 °C. The soluble acetic acid/urea plaque extracts were subjected to reverse phase HPLC using a 260 × 7-mm RP-300 Aquapore (Applied Biosciences Inc., Foster City, CA) column, eluted with a linear gradient of aqueous acetonitrile. Eluant was monitored continuously at 230 and 280 nm, and 1-ml fractions containing Mfp-3S were pooled and freeze-dried, injected into Shodex-803 column (5 μm, 8 × 300 mm), which was equilibrated and eluted with 5 % acetic acid in 0.1 % trifluoroacetic acid. Eluant was monitored at 280 nm. Sample purity was assessed by acid urea-PAGE, amino acid analysis, and MALDI time-of-flight mass spectrometry. Fractions with pure Mfp-3S were freezedried and redissolved in buffers for further studies. About 3 mg Mfp-3S can be purified from 1000 freshly (within 24 hrs) secreted plaques.

Zeta potential

Zeta potentials of Mfp-3S in solution (~0.1 mg/ml) were obtained using the Malvern Nano ZS which is calibrated regularly using Malvern Zeta Potential Transfer standard (P/N DTS1230, Batch number 380901). Zeta potentials of Mfp-3S were measured as + 23 mV, + 6 mV, and − 0.8 mV, respectively, in pH 5.5, 6.5 and 7.5 buffer with 100 mM ionic strength.

Mfp-3S self-coacervation and turbidity measurement

Stock solutions of 1 mg/ml Mfp-3S were prepared in 10 mM acetic acid buffer (pH 3). The final protein concentration was fixed at 0.1 mg/ml by adding stock solution to buffer at volume ratio of 1:9 (stock: buffer). Coacervation of Mfp-3S at different buffer conditions was measured turbidimetrically at 600 nm by UV-vis spectrophotometry. Mfp-3S absorbance was negligible at 600 nm. The relative turbidity is defined as ln (T/T0) where T and T0 are light transmittance with and without sample, respectively [21].

Microscopy

The turbidity associated with coacervate droplet formation was visually inspected by inverted light microscopy. Protein distribution was also investigated by using an Olympus DSU Fluorescent Microscopy (model IX81 DSU, Olympus, Tokyo, Japan). Images were taken with an ImagEM camera (C9100-13, Hamamatsu, Shizuoka, Japan) under the control of MetaMorph software (Olympus). Desired excitation and emission wavelength was obtained by mercury bulb combined with 89000 Sedat Quad Filter Set (Chroma Tech. Corp.)

Quantification of adsorbed coacervate by amino acid analysis

A similar structure to that used in microscopy was created using rectangular glass cover slides; double sided tape was used to stick two cover slides together along their margins. A 50~100 μl volume of Mfp-3S coacervate (0.1 mg/ml) was then injected into the gap and left undisturbed for 1 hr to let the coacervate adsorb or settle on glass surfaces. The upper and bottom glass slides were then separated and broken into pieces to fit in 1ml hydrolysis vials. 100 μl of 2 M HCl and 5 μl of phenol were added to the vials containing the glass samples, which were then vacuum sealed, followed by hydrolysis at 158 °C for 1 hr. After hydrolysis, the solutions were washed with twice with water and then twice more with methanol via flash evaporation. The hydrolyzed products were dissolved in 0.02 M HCl and a Hitachi L-8900 Amino Acid Analyzer for routine amino acid analysis.

Adhesion measurement by SFA

The adhesion of Mfp-3S coacervate on mica was measured by SFA. The details of the SFA technique have been described elsewhere [22]. The coacervate deposition was done by placing 40 μl of Mfp-3S coacervate (0.1 mg/ml) between two mica surfaces. After 1 hr settlement and absorption, the bottom mica surface were brought in contact with the upper one, and further compressed for another 1 min before separation All experiments were performed at room temperature thermostatted at 22 °C.

QCM-D

Gold sensors used were purchased from BiolinScientific (QSX301) and cleaned according to the protocol suggested before use. QCM-D experiments were carried out in a Q-Sense E4 system using two flow modules in parallel. Samples were introduced into the modules at a 0.1ml/min flow-rate using a 4-channel Ismatec IPC-N 4 peristaltic pump. In QCM-D, changes in resonance frequency (ΔF) and dissipation (ΔD) of a quartz crystal are recorded to measure the amount and viscoelastic property of the material deposited onto the sensor respectively. The crystal is excited at its fundamental frequency, approximately 5MHz, and changes can be observed at the fundamental (n=1) as well as overtone frequencies (n=3, 5, 7, 9, 11). Readings taken at the fundamental frequency are not usually used as they are prone to artifacts from the sensor clamp.

Results and Discussion

Characterization of Mfp-3S coacervate morphology

Coacervation is typically measured by turbidimetry as turbidity increases when macromolecules associate to form phase-separated fluidic droplets. Protein precipitation also leads to turbidity but the droplet morphology of coacervated macromolecular aggregates is easily distinguished from precipitates by light microscopy (Figure 2). For microscopic observation of coacervation and the coalescence of droplets, Mfp-3S coacervates were pre-formed in pH 5.5 acetate buffer, then injected into a gap between two glass slides.

Figure 2.

Figure 2

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Visualization of coacervation using light microscopy and spectrometry. (a, b) Microscope images of Mfp-3S coacervates taken 5 min (a) and 25 min (b, c) after injecting samples into cells for imaging (a, b: bright field; c: fluorescence). The 5 min time point was selected on the basis of convenience as it was easily obtained after injecting the coacervate between the glass slides. The 25 min time point was chosen as the second time point as there is no discernible change in the adsorbed coacervate droplets after 25 min; (d, e) enlargement of parts of (a) and (b) respectively; (f) comparison of Mfp-3S in dispersed solution (left) and associated coacervate (right).

Figure 2 (Panels a, b, d, e) shows bright field microscope images of coacervate droplets that have settled onto the bottom slide. As time elapsed, more coacervates settled and adsorbed onto the bottom surface with a lesser amount also adsorbed to the upper surface. By using amino acid analysis to quantify coacervate deposition on the two surfaces (upper/lower ratio: 0.25 ~ 0.20, Figure 3) following bulk depletion, we estimated how readily the coacervates adsorb to glass (upper surface) relative to gravity-dependent droplet settlement (lower surface). Visualizing macromolecules in coacervates usually requires functionalization with molecular, often fluorescent, probes, however, this is unnecessary with Mfp-3S as it contains 10 mol% tryptophan (Trp) that imparts an intense intrinsic fluorescence. With UV excitation (Figure 2c), Mfp-3S derived coacervates are readily visualized under microscopic observation.

Figure 3.

Figure 3

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(a) Schematic comparison of coacervate yields on upper and lower surfaces, which are caused by adsorption only, and the combination of adsorption and settlement, respectively. (b) amino acid analysis results of 3 pairs of samples.

The effect of buffer pH, ionic strength, temperature on coacervation

The turbidity of the Mfp-3S dispersions was measured to quantify the yield of coacervate under different buffer conditions. Figure 4a shows the data collected one minute after mixing protein stock and buffers. In the range of pH and ionic strengths tested (avoiding those pH regimes where Dopa residues are highly vulnerable to autoxidation), the turbidity was found to increase with pH and ionic strength; and above a certain ‘critical’ pH or ionic strength, the protein precipitated from the solution. Given the apparent pI ~ 7.5 from zeta potential measurements (compared with a predicted pI ~8 ExPAsy) for Mfp-3S, the protein is well dispersed in buffer at pH 3 and low monovalent salt concentrations (~10 mM) due to the long-range electric double-layer repulsion between the net positively charged molecules. At high ionic strength, electrostatic ‘double-layer’ repulsion is largely screened. As the buffering pH is increased and approahes the pI ~ 7.5 (the pH at which the positive and negative charges exposed on molecule’s surfaces exactly neutralize each other), the net charge of the Mfp-3S molecules decreases to zero, with a corresponding decrease and eventual disappearance of the long-range double-layer repulsion. Any two contacting droplet surfaces now expose an equal number of positive and negative charges, and coulombic interactions can form between the two surfaces resulting in strong intermolecular attraction. In summary, increasing both the pH and ionic strength leads to a decreasing long-range repulsion and increasing short-range attraction (binding adhesion) that results in the coalescence of the soluble proteins, first as coacervates and, then, as precipitates.

Figure 4.

Figure 4

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Dependence of coacervate-associated turbidity on pH and time. Turbidity of 0.1 mg/ml Mfp-3S in different buffer conditions. Buffer 1: phosphate buffer; 2: acetate buffer; 3: phosphate buffer.

Given the observed dependence on pH and ionic strength, electrostatic interactions definitely contribute to Mfp-3S coacervation. Figure 4b shows turbidity changes with time. Upon suspension in aqueous solution with pH and ionic strength suitable for coacervation, the Mfp-3S molecules initially phase separate as coacervate droplets. As the droplet size increases, so does the turbidity of the solution, peaking at around 30min. However, with additional time, the coacervate droplets settle onto the surface of the enclosure due to their higher density. In addition, the low interfacial energy causes the droplets to spread out upon contact with any surfaces. The decrease in turbidity after 30min thus reflects bulk depletion of the coacervate droplets by sedimentation and surface adherence rather than resolubilization of the Mfp-3S coacervates.

The influence of temperature on Mfp-3S coacervation was also examined. As shown in Table 1, turbidity decreases dramatically with temperature T. Previous studies have determined that T affects coacervation according to the driving forces involved: in electrostatically-driven complex coacervation, increasing T typically decreases turbidity due to the weaker attraction with increasing T [23], whereas for hydrophobically-driven coacervates such as elastin, raising T leads to higher turbidity due to the entropy-driven association of the molecules [18]. As such, the changes in turbidity reflect the net energy balance of these different trends. For this study, the decrease in electrostatic interaction at higher T is such that they appear to overcome the entropy gain at higher T to result in decreased turbidity.

Table 1.

Mfp-3S (0.1 mg/ml) coacervate turbidity change along with temperature.

Turbidity change with T Room T 18 °C 37 °C 50 °C 70 °C 80°C
pH 5.3 10 mM 14 3 – – –
pH 6.2 100 mM 86 80 61 36 27
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Mfp-3S is the most hydrophobic among all known mussel adhesive proteins with at least 60% of the amino acid residues in the sequence being more hydrophobic than glycine [17]. The contributions of hydrophobic interactions to the coacervation of Mfp-3S must thus be considered. Figure 5 compares macromolecular interactions in three different coacervating systems: 1) typical polycation/polyanion complex coacervation, 2) Mfp-3S, and 3) tropoelastin single component coacervation. The optimum pH for complex coacervation by oppositely charged polyelectrolytes, is where the polyanions and polycations (such as gelatin and gum Arabic) neutralize one another and the intermolecular electrostatic attraction is the strongest. In contrast, as pH increases, long-range repulsion between Mfp-3S is only partially overcome (complete internal neutralization occurs only when the N-terminus loses a H+ at ≥ pH 7.5). Even before reaching pH 7.5, the Mfp-3S proteins tend to cluster more freely and may even rearrange to facilitate short-range coupling of +/− charges to achieve localized charge neutralization. Given that Mfp-3S coacervation is observed at pH < pI (Figure 4a), protein coalescence cannot depend exclusively on the electrostatic interactions between the zwitterions, but must also include hydrophobic interactions between the molecules’ significant hydrophobic domains. However, the hydrophobic interactions do not contribute to Mfp-3S coacervation in the same way as they do in the coacervation of elastin which exhibits increased coacervation with increasing T [24] – a trend that is opposite to that of Mfp-3S as discussed earlier. In tropoelastin, increasing T is essential for the entropy-driven aggregation of hydrophobic domains that simultaneously excludes the positive charges from the hydrophobic core (Figure. 5). There are no other reports of single proteins that phase-separate by coacervation; indeed, proteins typically precipitate when pH = pI. However, coacervation by zwitterionic gemini surfactants has been observed [26]. These surfactants are, in a way, miniatures of Mfp-3S in having both positive and negative charges separated by a neutral core domain and end with nonpolar hydrocarbon tails [25]. These features were shown to be critical for coacervation hence corroborate our model for Mfp-3S coacervation.

Figure 5.

Figure 5

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A scheme comparing three different coacervation systems: one complex coacervation and two single-component coacervations. The coacervation of oppositely charged polyelectrolytes (left) occurs at the pH condition where the net charge of the complex is neutral. In Mfp-3S (middle), increasing pH deprotonates the carboxylic group of Asp and C-terminus (pKa ~ 4) resulting in a zwitterion. Driven by intermolecular charge coupling and hydrophobic interactions between hydrophobic domains, the zwitterionic protein asscoaciate to form coacervates. Hydrophobic interaction is responsible for overcoming the intermolecular electrostatic repulsion caused by the extra positive charge when pH is lower than Mfp-3s pI. In contrast, the self-coacervation of tropoelastin (pI>10) at any given pH is induced by increasing the temperature. Note that Lys charges (⊕) are excluded from the coacervate and neutralized by microions at the surface [35]. Molecules are not drawn to scale. The blue background represents the coacervate defined by the macromolecule complex, the shape of which is arbitrary.

Interfacial energy and wettability of Mfp-3S coacervate

Coacervates with coating or adhesive functions should exhibit low interfacial energy or tension. The adhesive capillary force of Mfp-3S coacervate was measured by SFA as follows: coacervate preformed in buffer at pH 5.5 was injected in between two well-separated mica surfaces, and given 1 hr to equilibrate, adsorb, and coalesce (spread) on the mica surfaces before performing any measurements. Then, the lower mica surface was brought up into contact with the upper surface, and further compressed for 1 min to allow the coacervate layers on the two surfaces to coalesce, forming a capillary bridge or neck. The bumpy fringe shown in Figure 6a is an indication of the rough surface and heterogeneous structure/morphology of the coacervate layers and the bridging neck. Upon separation, normalized ‘separation forces’ (also ‘pull-off’ or ‘adhesion’ forces) ranging from F/R = −8 to −20 mN/m were measured depending on the pulling rate (5 to 35 nm/s). The effective interfacial energy, γeff, can then be deduced from the measured adhesion forces, F, and the radius of curvature, R, using [7, 26]

Figure 6.

Figure 6

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(a) Schematic of the SFA adhesion experiment and corresponding FECO patterns in each step ; (b) a representative force run plot with 35 nm/s pulling rate.

γeff=F/3R (1)

The interfacial energy γeff was calculated to range from 0.5–3.7 mJ/m2. Such a low interfacial energy is consistent with that of other coacervates [7, 27] and of great importance to adhesion since the ability to spontaneously wet and spread over a surface is the hallmark of a good adhesive and reliant on low interfacial tension. Another demonstration of good wettability is the ‘anti-coffee ring’ effect [28] shown by Mfp-3S coacervate (Figure 7), where instead of forming a ring-like deposit along the perimeter of air-dried droplets (as Mfp-3S does in solution), Mfp-3S coacervate uniformly stains the glass substrate. Given that many applications in printing [29], biology [30], and complex assembly [31] require uniform coatings, Mfp-3S coacervates represent a new class of ‘complex fluid’ coating materials.

Figure 7.

Figure 7

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Microscope images of ‘coffee stain’ left by 10 μl of Mfp-3S coacervate (a, b) and solution (c, d), which show ‘anti-coffee ring’ and ‘coffee ring’ effects respectively. The ‘anti-coffee ring’ effect of Mfp-3S coacervate indicates its uniform wetting capability.

Adsorption of Mfp-3S coacervate on HAP by QCM-D

HAP is a bioceramic analogue of the mineral component of human bone and teeth. Understanding the interaction between coacervates made of Dopa-rich Mfps and HAP surface could inspire the design of improved medical adhesive and implant surfaces. The quartz crystal microbalance-dissipation (QCM-D) was used here to investigate the adsorption of Mfp-3S coacervates on HAP surfaces, which was also compared to the adsorption performance of Mfp-3S in solution and lysozyme (Figure 8). Mfp-3S in solution or in coacervated form was obtained by using different buffer conditions. Given that the frequency change ΔF is proportional to mass change, it is clear that the adsorbed amount of lysozyme is the lowest among all the tested samples. Mfp-3S solution shows higher adsorption on HAP than lysozyme, but much less than coacervated Mfp-3S.

Figure 8.

Figure 8

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(a) Changes of frequency in QCM-D experiments after exposing HAP surface to Mfp-3S solution (pH 3, 10mM); coacervate (pH 3 100mM, pH 5.5 100mM) and lysozyme solution in the above 3 conditions. (b) Plots of ΔD vs ΔF. The higher ΔD/ΔF value is, the softer the material is.

Two coacervate samples prepared under different conditions were tested: pH 5.5/ionic strength 100 mM (the ‘optimized’ coacervate, i.e., optimum conditions for Mfp-3S coacervate as indicated by turbidity measurement), and pH 3/ionic strength 100 mM (‘non-optimized’ coacervate, sub-optimal conditions). As expected (Figure 8a), the optimized coacervates exhibited better adsorption than non-optimized ones. After rinsing with buffer, the mass loss of optimized coacervate was only 20% compared with a 75% loss in the suboptimal coacervate. Based on these results, coacervated Mfp-3S demonstrated excellent adsorption on HAP, primarily based on electrostatic and H-bonding interactions between the protein and HAP, and also the interactions between the proteins themselves, which drive the continuous build-up of protein on top of the first protein layer adsorbed to the HAP surface. The interaction between protein and HAP is likely to be mainly H-bonding between the protein Dopa and the phosphate groups on HAP, enhanced by the electrostatic interaction between the positively charged protein and net negatively charged HAP surface. It is expected that Dopa/phosphate H-bonding will be weaker at pH 5.5 than at pH 3 due to Dopa autoxidation [32, 33]. The adsorption of lysozyme (pI ~11) on HAP appeared independent of the buffer pH used, which is consistent with electrostatic interactions between phosphate groups and lysine and arginine, whose charges would not change in the pH range tested given reported pKas of 10.4 and 12.5, respectively [34]. Considering that both lysozyme and Mfp-3S are basic, electrostatic interactions between Mfp-3S and HAP at all three buffer conditions is unlikely to be the major reason for the differences in adsorption. From these results, it can thus be inferred that the highest adsorption of Mfp-3S ‘optimized’ coacervate, and lowest mass loss during rinsing (compared with non-optimized coacervate) are due to the same strong intermolecular interactions (cohesion) that also drive protein coacervation. The dissipative change ΔD is an indication of a material’s viscoelastic properties. The higher the ΔD, the more fluidic, or, ‘softer’ the material is; the lower the ΔD, the more solid, or, ‘stiffer’ the material is. It is clear from Figure 6b that the absorbed layer of coacervate is the most fluidic of all the tested samples, to the extent that its changes in ΔD represent changes in viscosity. The significant hysteresis exhibited by the coacervate suggests that it may be ideally suited for dissipating energy associated with deformation of the adhesive plaque produced by drag and lift forces.

Conclusion

Mfp-3S is the first known naturally occurring self-coacervating adhesive protein from the mussel and, along with tropoelastin, the only proteins known to self-coacervate. In marked contrast to elastin coacervation, which is hydrophobically driven, the phase separation of Mfp-3S is markedly dependent on ionic strength and pH – the hallmarks of complex coacervation - but, unlike the coacervation of gelatin and gum Arabic, for example, is optimal at pH values below those necessary for protein charge neutralization. Conditions for Mfp-3S coacervation are perfectly adapted for the solution conditions that exist under the foot during plaque formation, namely an acidic pH at ~0.1 M ionic strength [33]. Electrostatic and hydrophobic interactions between and within Mfp-3S under these conditions drive the association of protein molecules to form a fluid phase that is separate from bulk water. One-component coacervates formed by Mfp-3S may circumvent much of the instability and complicated solution chemistry associated with binary and ternary coacervates. Given the low interfacial energy of coacervated Mfp-3S and its superior adsorption on HAP surfaces shown by the SFA and QCM-D experiments, respectively, it is highly likely that the coacervates formed from recombinant Mfp-3S or its synthetic analogues can be used in future investigations to explore potential dental or orthopedic adhesive applications.

Acknowledgments

This research was supported by MRSEC Program of the National Science Foundation under the award No. DMR 1121053 and by NIH grant No. R01 DE018468. We thank Dr. Matthew Dixon (Biolin Scientific) and Dr. Mary Raven (NRI/MCDB microscopy facility, UCSB) for their help with QCM-D measurements and microscope training respectively.

Footnotes

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