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Nanopore Diameters Tune Strain in Extruded Fibronectin Fibers Mohammad Raoufi, Tamal Das, Ingmar Schön, Viola Vogel, Dorothea Brüggemann, and Joachim P. Spatz Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.5b01356 • Publication Date (Web): 11 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Nanopore Diameters Tune Strain in Extruded Fibronectin Fibers Mohammad Raoufi +,1,2,3, Tamal Das+1,2, Ingmar Schön4, Viola Vogel4, Dorothea Brüggemann*1,2, Joachim P. Spatz*1,2 1

Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems,

Heisenbergstr. 3, D-70569 Stuttgart, Germany 2

Department of Biophysical Chemistry, University of Heidelberg, INF 253, D-69120

Heidelberg, Germany 3

Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical

Science, Tehran 1417614411, Iran 4

Laboratory of Applied Mechanobiology, Department of Health Sciences and Technology

ETH Zurich, Vladimir-Prelog Weg 4 (HCI F443), CH-8093 Zurich, Switzerland

* To whom correspondence should be addressed: [email protected], [email protected] + Authors contributed equally to this work

Abstract Fibronectin is present in the extracellular matrix and can be assembled into nanofibers in vivo by undergoing conformational changes. Here, we present a novel approach to prepare fibronectin nanofibers under physiological conditions using an extrusion approach through nanoporous aluminium oxide membranes. This one-step process can prepare nanofiber bundles up to millimeter length and with uniform fiber diameters in the nanometer range. Most importantly, by using different pore diameters and protein concentrations in the extrusion process, we could induce varying lasting structural changes in the fibers, which were monitored by fluorescence resonance energy transfer and should impose different physiological functions.

Keywords Conformational changes, Protein nanofiber, AAO template, FRET, Extrusion

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Introduction Cell fate is affected by the geometrical size and mechanical properties of protein fibers in the extracellular matrix (ECM), especially in the case of collagen1-3 and fibronectin (FN)4, 5. Such native FN fibers span a diameter range of a few nanometers up to micrometers in vivo6, 7. The in vivo fibrillogenesis of FN is initiated by binding to cell surface receptors, such as adhesionmediating integrins, and the application of forces, which partly unfold FN 7, 8. In this process specific epitopes of FN are presented to the cell membrane, which direct subsequent cell signaling events. Hence, switching epitope exposure by stretching ECM protein fibers can directly influence cell fate by activating certain signaling cascades 9, 10. For tissue engineering applications and to understand the underlying principles of native FN fibrillogenesis and its functionality in context with cells in more detail, it is required to prepare FN fibers with defined conformations under physiological conditions and in a reproducible manner. Several approaches to study FN fibrillogenesis in a cell-free environment have been introduced over the past years 11-25. Fiber assembly via mechanical pulling of single FN fibers from a drop of protein solution was first introduced in the 1990s by Brown and co-workers 11, 13, 17, 25

. This shear force-induced method yielded FN fibers in the range of several

micrometers in diameter. This fiber pulling method was then employed to probe stretchinduced alterations in the structure-function relationship using fluorescence resonance energy transfer (FRET)

6, 19

combined with binding assays 6, 15, 20. Alternatively, fibrillar networks of

FN with fiber diameters in the micron range were also formed by a dynamic self-assembly process underneath lipid monolayers 12. Moreover, surface-induced fibrillogenesis of FN on hydrophobic substrates 23 and negatively charged surfaces 22 were introduced. These materialdriven methods yielded FN fibers between 10 and 100 nm. Recently, the combined effect of flow- and surface-induced conformational changes was used to analyze FN fibrillogenesis on stainless steel in a concentration-dependent manner

26

. Another concept to study


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fibrillogenesis in vitro is the preparation of FN nanofibers by de-wetting of a protein solution on hydrophobic silicon micropillars 18, 24. In this process the pillar geometry could be used to adjust the fiber diameter between 20 and 160 nm. Finally, nanofabrics were formed from micropatterned FN ribbons, which were stamped onto a polymeric substrate. Upon substrate dissociation, the FN textiles folded into free-standing fibrillar arrays of up to 10 nm thickness 4, 16

.

While all the mentioned approaches are rather unique to fibronectin, other methods to prepare protein nanofibers have emerged from the growing area of tissue engineering electrospinning

32

, molecular self-assembly

33

, and phase separation

34

27-31

, including

. The latter two

approaches allow the formation of protein fibers with variable mechanical strength, yet both methods fail in terms of scalability, continuity, and fiber yield

31, 35

. Electrospinning is

presumably the most common process for nanofiber generation. It is inexpensive, continuous, scalable and allows a broad range of fiber diameters from a few nanometers to several micrometers. The process is, however, limited by the instability of the fluid jet and the toxic solvents, which often change the native conformations of the protein molecules

35, 36

. Most

importantly, electrospinning approaches have not yet succeeded in the preparation of FN nanofibers: only synthetic polymers or other protein nanofibers with FN surface modifications were presented to date 36-38. In many of the previous fibrillogenesis model systems, FN fibers were pulled from an aqueous phase into air

6, 19, 39

, which affects their ultrastructure

6, 19, 39

. Here we introduce a

new process, involving extrusion of protein solutions through nanopores, which is capable of preparing FN nanofibers with reproducible diameters under physiological conditions. As the proof-of-concept, we present the water-based extrusion of FN nanofibers without the protein ever coming in contact with air or another solvent. The process is simple, one-step, inexpensive and can produce nanofiber bundles up to millimeter length. By tailoring the nanopore diameter and the protein concentration, we were able to control the conformation of FN molecules in the extruded fibers and could thus establish a new model system for in vitro studies on FN fibrillogenesis.

Experimental FN was purified from human plasma by gel filtration and affinity chromatography as previously described6, 19 and FRET-labeled FN was prepared using an established procedure 40, 41

. As determined by absorption measurements6, FN was labeled with 6.2 donors (Alexa

Fluor® 488, labeled on Lys) and 3.6 acceptors (Alexa Fluor® 546 labeled on Cys) on average. For the fabrication of FN nanofibers, FN was dissolved in PBS (Life Technologies, Darmstadt, Germany) to final concentrations ranging from 100 µg/ml to 1000 µg/ml. 5% of labeled FN was mixed with 95% of unlabeled FN. This ratio was chosen to prevent


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intermolecular FRET signals during the fluorescence intensity measurements 6. The FN nanofibers were fabricated by manual extrusion into an aqueous buffer through anodized aluminium oxide (AAO) nanopores in a customized setup (see fig. 1). Nanoporous AAO membranes with pore diameters dAAO of 20 and 70 nm were prepared by anodization of aluminum foils in a home-built setup according to our previous work

42

. Commercial

Whatman® Anodisc membranes with a pore diameters of 200 nm were purchased from Sigma Aldrich (Munich, Germany). Under manual setting, we did not observe any significant variation in this extrusion flow rate (Qe) for different pore diameters (dp = 20, 70, and 200 nm) for an aqueous buffer, which did not contain any protein molecules. In all cases, we found Qe to fall between 250 ± 50 µl/min. We then estimated the flow rate (Qp) and mean flow velocity (Vp) for a single pore for each pore diameter value. For this purpose, we first calculated the average number of pores (Np) in a membrane by multiplying the total membrane area (49 mm2) by the porosity of each membrane and dividing by the mean pore cross-section area (Ap). The porosity was known to be 0.15, 0.16, and 0.1 for 20, 70, and 200 nm pores, respectively. We consequently determined Qp and Vp as: Qp = Qe/ Np and Vp = Qp/Ap. We found Qp = 0.01 ± 0.002, 0.12 ± 0.024, and 1.60 ± 0.32 pl/min and Vp = 0.57 ± 0.11, 0.53 ± 0.11, and 0.85 ± 0.17 mm/s for 20, 70, and 200 nm pores, respectively. The extruded nanofibers were collected on a glass substrate (Gerhard Menzel GmbH, Braunschweig, Germany) and analyzed in PBS solution using a confocal fluorescence microscope (Leica TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany). The samples were excited at 488 nm, and the two emission detection windows were set at 495-545nm (donor channel) and 565-635nm (acceptor channel) to capture the peak emissions. Reference measurements of free FN in solution were performed with a concentration of 500 µg/ml containing 40% of labeled and 60% of unlabeled FN. Denatured protein solutions were prepared with 4 M of guanidine hydrochloride (GdnHCl, Sigma Aldrich, Munich, Germany), and for molecular crowding experiments we used dextran 70 from Carl Roth GmbH (Karlsruhe, Germany). We used a Matlab script (Version 8.2) to analyze the FRET signals obtained from extruded FN nanofibers in PBS. The FRET intensity ratio of the acceptor channel and the donor channel (IA/ID) was calculated for each pixel within the field of view and displayed as histograms. The script subtracted the dark current background from each channel (acquired from each experiment). Then we applied a Gaussian blur (with a 1-pixel standard deviation) to each channel to smoothen the intensity value. The intensity of the 12bit acceptor channel for each pixel was then divided by the intensity of the corresponding 12bit donor channel to yield the IA/ID ratios. Subsequently, the FN nanofibers were prepared for scanning electron microscopy (SEM) analysis and rinsed with PBS to be dried at room temperature. The dried nanofibers were


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coated with approximately 7 nm gold and analyzed using a Zeiss Ultra 55cv device (Zeiss, Oberkochen, Germany). All measurements were performed with an operation voltage of 5 kV. The software ImageJ (1.44p) was used to analyze the SEM images. We statistically measured the average fiber diameters from at least 30 fibers per image (± 2σ).

Results and discussion AAO membranes were fabricated in a low-cost anodisation process, which yielded highly ordered, self-organised nanopores with regular pore shapes and uniform size distributions as previously presented

42-45

. These vertical nanochannels with uncharged pore walls were then

employed to prepare FN nanofibers in PBS buffer by extrusion through the AAO nanopores (see Fig 1). This simple and easy-to-use method allowed us to manually produce a new class of protein nanofibers in a proof-of-concept approach. Moreover, with our novel extrusion approach we prepared FN nanofibers with significantly increased yield compared to the currently used phase separation or molecular self-assembly approaches

33, 34

. During the

preparation of FN nanofibers the concentration of the protein feed solution was varied from 100 µg/ml to 1000 µg/ml, and the nanopore diameter was changed between 20 nm and 200 nm. The FN fibers, which were extruded onto glass slides and dried in air prior to SEM analysis, either assembled into micron-sized bundles of nanofibers (see Fig. 2A) or arranged into large-scale nanofibrous meshes (see Fig 2B). Single nanofibers (see Fig. 2C) were also found to align with neighboring nanofibers (see Fig. 2D). The diameter of single extruded FN nanofibers was found to change reproducibly in dependence of the extrusion parameters. For the smallest AAO pore diameter of 20 nm the fiber diameter was around 17 ± 3 nm for all concentrations. With 70 nm large nanopores the fiber diameter changed from 61 ± 3 nm for 100 µg/ml to 66 ± 5 nm 1000 µg/ml. With the largest pore size of 200 nm the fiber diameter increased more distinctively from 57 ± 7 nm for 100 µg/ml to 152 ± 12 nm for 1000 µg/ml. Hence, the diameter range of single extruded FN nanofibers and nanofiber bundles was close to the diameters of native FN fibers, which can vary from only 10 nm up to 1000 nm 7, 46.


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A Feed solution Mounted AAO membrane

Substrate

B

200 nm

C

D

1 m

Fig. 1: Extrusion of protein solution through a nanoporous AAO membrane. (A) Schematic drawing of the extrusion setup: the protein solution is pushed through the mounted AAO membrane and collected on a glass substrate, (B) SEM image of AAO nanopores in top view, (C) Cross-sectional SEM image of AAO nanopores, (D) Schematic cross-sectional view of protein solution (red) being extruded through a single AAO nanopore by applying pressure from the top. Confinement to the nanopores induces FN fibrillogenesis and the resulting Fn nanofibers are extruded into a receiving chamber filled with PBS on the substrate placed below.


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Fig. 2: SEM images of FN nanofibers, which were extruded into an aqueous phase with different concentrations and nanopore diameters dAAO and deposited onto glass slides: (A) micron-sized bundle of FN nanofibers (c = 500 µg/ml, dAAO = 70 nm), (B) randomly oriented mesh of FN nanofibers (c = 500 µg/ml, dAAO = 70 nm), (C) single FN nanofiber (c = 1000 µg/ml, dAAO = 200 nm) and (D) aligned FN nanofibers (c = 1000 µg/ml, dAAO = 200 nm). To correlate the parameters in our manual extrusion process with the molecular-level structural changes in the resulting FN nanofibers, we carried out a FRET analysis of nanofibers in PBS. Prior to extrusion, double-labeled FN was mixed with unlabeled FN at a ratio of 1:19 to prevent intermolecular FRET. Fig. 3 shows representative confocal fluorescence microscopy images of donor, acceptor and the FRET index IA/ID of FN nanofibers, which were extruded with two different sets of parameters. The structure of the fibrous assemblies in PBS was stable for several hours.


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Fig 3: Confocal fluorescence microscopy images of extruded FN nanofibers containing double-labelled FRET-FN. The FN fibers were extruded with different parameters and the resulting conformation of FN did not change as a function of time: (A) donor, (B) acceptor, (C) FRET index IA/ID of FN nanofibers extruded with c = 1000 µg/ml and dAAO = 20 nm, (D) donor, (E) acceptor, (F) FRET index IA/ID of FN fibers extruded with c = 500 µg/ml, dAAO = 70 nm. Varying FRET indices along individual fiber bundles in (C) and (F) indicate that different unfolding regions have been induced by pulsatile flow conditions in our manual extrusion process.

For different extrusion parameters, the corresponding FRET histograms are shown in Figure 4. The width of the histograms reflects the pixel-to-pixel variations of the local averaged FN intensity, including detection noise of the fluorescence microscope 41. From the representative histograms shown in Fig 4A and B it is evident that decreasing AAO nanopore diameters, and reduced concentrations of the protein feed solutions resulted in lower mean values of the corresponding FRET indices, i.e. more extended molecules. In comparison, the FRET ratio was 0.5 for the folded protein in solution (dimeric FN in PBS at a concentration of 500 µg/ml), and 0.19 for the denatured, i.e. unfolded, protein in solution (4 M GdnHCl in PBS). Previous solution measurements revealed that Fn starts to lose secondary structure at a 1 M GdnHCl concentration

41, 47

, and we obtained a FRET index of 0.25 for free FN with 1M

GdnHCl in our study. These reference measurements of FN in different solutions are indicated by black dashed lines in Fig. 4C. The FRET index mean values of extruded FN nanofibers in PBS, which were read out from the histograms, were in the range of 0.18 to


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0.41 and thus between the two reference measurements for folded FN and denatured and thus unfolded FN in solution (see Fig. 4C). The lowest FRET index of 0.18 was measured for nanofibers extruded through the smallest 20 nm pores at the lowest tested FN concentration (100 µg/ml). As this FRET index coincides with the FRET index of 0.19 from the denatured protein solution, this suggests that these particular FN nanofibers contained partially denatured FN.

When comparing the effects of pore diameter and FN concentration on the molecular strain of FN molecules within extruded nanofibers, we observed two prominent trends (Fig. 4A and B): First, decreasing the pore diameter progressively decreased the mean FRET index suggesting that FN gets progressively more stretched (Fig. 4A). This is a remarkable finding and agrees with other examples where proteins were reported to get unfolded during translocation of proteins through confined nanopores.48 This process is multistep, much more complicated than chemical denaturation of proteins in bulk and influenced by several physicochemical factors such as conformational entropy, protein-wall ionic interaction, protein-wall steric interaction, hydrodynamic interaction, and the driving force.48, 49 In order to pass through the confinement, a protein molecule must decrease the conformational entropy, and stretching becomes imminent.

50-52

All these effects should be very pronounced

when the gyration radius (Rg) of the protein molecule is comparable to the radius of the nanopore. Using light scattering, the gyration radius of native human plasma FN in its compact conformation was previously determined and it was estimated that dimeric FN is disk-shaped and has a diameter and thickness of 30 and 2 nm, respectively.

53

Hence, one

should indeed expect structural extension of FN in the confined nanoporous membrane like we observed, for the smallest pore diameter of 20 nm (i.e. 10 nm pore radius, see Fig. 4A and C). Although the hydrodynamic extensional or elongational forces, which account for the observed structural changes in the FN fibers, are strongest inside the confined nanopores of the AAO membrane, we assume that FN molecules might already experience moderate elongational forces before they enter the nanopores. These forces might start to extend the disk-shaped quaternary FN structure before the molecules enter the AAO nanopores, where they subsequently undergo a much stronger unfolding. The contribution of these waterinduced elongational forces outside the pores is difficult to separate from the influence of the hydrodynamic elongational forces inside the nanoporous membrane.

Second, increasing the bulk protein concentration increased the FRET index for all pore diameters (Fig. 4B and C). This implies that FN molecules are less stretched when extruded from more highly concentrated solutions. Using SEM analysis we observed that increasing


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protein concentrations resulted in nanofiber diameters reaching the dimension of the template pores. Accordingly, low protein concentrations of 100 µg/ml yielded fiber diameters that stayed below the template diameter and could not always be detected using confocal fluorescence microscopy and have therefore not been included in the analysis of the FRET index (see Fig. 4C). It is important to note here that in addition to the inherent protein structure or internal chemistry, the folding process also depends on the entropy, which is in turn governed by the excluded volume constraints. In the present scenario, at least two major factors influence this constraint: the pore diameter (confinement) and the protein concentration (crowding). Both of these factors are known to control the landscape of accessible conformations during coil-globule, crystallization, and demixing transitions. For example, previous simulation studies investigating the effect of macromolecular crowding on the conformation of polymer chain (which do not contain secondary structure) within tubelike confined environment have shown that a crowded environment, which depletes the accessible volume of the polymer molecule, counters the confinement-induced stretching forces54. This effect of crowding is reflected by a progressive shift of the mean radius of gyration towards lower values and a change in conformational scaling behavior, with increasing degree of crowding.

To elucidate whether molecular crowding results in less unfolding, i.e. higher FRET indices, due to the decrease of Rg, we have also performed extrusion of 250 µg/ml FN solutions in the presence of dextran 70. In previous studies on protein folding and aggregation, this wellestablished crowding agent was used in physiologically relevant concentrations up to 200 mg/ml 55-57. Therefore, we added 100 mg/ml of dextran 70 to the FN solution prior to extrusion and obtained a FRET index of 0.31 ± 0.03, compared to only 0.28 ± 0.02 in PBS. When 150 mg/ml dextran 70 was used as crowding agent, the FRET index even increased to 0.32 ± 0.03 (see black star in Fig. 4C; p-value = 4.6 × 10-6; student’s t-test). Macromolecular crowders generally facilitate the association of proteins through volume exclusion effects and favor more compact states 55-60. The increase in FRET index in presence of dextran 70 is thus in agreement with the observations made on in vitro systems 55-57. It is interesting to note here that in vitro crowding-mediated shifting of reactions to the folded state is most effective when the molecule is in transition between folded and unfolded states 55. In vivo macromolecular crowding dominates the extracellular microenvironment, and a study investigating its effect on extracellular matrix organization has previously shown that crowding promotes supramolecular assembly and alignment of extracellular matrix proteins, affecting proliferation, adhesion, and migration behavior of mesenchymal stem cells 61. What is nonintuitive here is the dominance of the crowding effect even in the presence of high fluid elongational forces, in addition to confinement-induced stretching forces. The water-induced


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extension may completely distort the energy landscape, opening otherwise cryptic intermolecular binding sites and thus, accelerating their bundling in a concentration dependent way 62. However, when pores are so small that the pore radius (10 nm) is in the range of the 2 nm thickness of dimeric FN, only very few FN molecules pass through the membrane. Hence, for the smallest pores, we observed relatively less conspicuous increase in the FRET index with the increasing bulk concentration as compared to that for other pore values (Fig. 4C). It is interesting to note that our FRET observation clearly supports this notion (Fig. 4C).


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Fig 4: Histograms of the FRET index measured from FN nanofibers using confocal fluorescence microscopy. The histograms in (A) and (B) represent IA/ID data collected from pixels of the fluorescence images that contained FN nanofibers. (A) At a concentration of 1000 µg/ml the pore diameter was varied from 20 to 200 nm. With increasing pore diameter the maximum of the FRET index histograms was shifted towards higher values. (B) For a


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constant pore diameter of 20 nm the maximum of the FRET histograms increased when the protein concentration was increased from 100 µg/ml to 1000 µg/ml. (C) The average FRET index IA/ID and its standard error from 2-3 confocal images is shown in dependence of the protein concentration and the AAO pore diameter. Blue line represents 200 nm pores, red line 70 nm pores and green line 20 nm pores. The black lines at the FRET indices 0.50, 0.25 and 0.19 indicate reference measurements of free FN in PBS, FN in PBS with 1M GdnHCl and FN in PBS with 4M GdnHCl, respectively. The black star at the 250 µg/ml FN concentration represents the FRET index in the presence of 150 mg/ml of the molecular crowder dextran 70.

Our results underline that the new method of extrusion-driven fibrillogenesis induced varying degrees of unfolding in the FN nanofibers due to the applied external force

63

. Decreasing

IA/ID ratios were caused by increased molecular extension and domain unfolding in the FN nanofibers

40

. We were able to reproducibly vary the observed conformational changes by

adjusting the concentration of the protein feed solution and the AAO nanopore diameter in the extrusion process. With this well-controllable, yet proof-of-principle, method to fabricate protein nanofibers under physiological conditions we have introduced a new model system to study FN fibrillogenesis in a non-cellular environment. Compared to existing approaches for in vitro fibrillogenesis, our novel extrusion approach has several advantages. Primarily, it is possible to study fibrillogenesis in aqueous environments for different fibrous assemblies with a very high fiber yield and unique diameter range, from only 15 nm for single nanofibers to several micrometers for fiber bundles. Previous studies, which used manual fiber pulling 6, 17, 19

, surface-induced fibrillogenesis

wetting on silicon micropillars

18

23, 24

, self-assembly under lipid monolayers

12

or de-

to analyze fibrillogenesis in vitro, were either restricted by

the fiber yield, the lack of diameter control and range or the fact that the fibers were attached to underlying pillar substrates. Furthermore, extrusion-induced fibrillogenesis could be performed at various temperatures, which do not impede the protein function. Most importantly, the presented extrusion approach could be used to study FN fibrillogenesis in vitro in an aqueous-based model system, which prevents solvent-induced conformational changes within the protein 64, and is much closer to in vivo fibrillogenesis than fiber pulling at the air-liquid interface 6, 19, 39. In future studies our novel approach can be developed further to a allow for fully-automated extrusion with precise flow control, which will facilitate the highthroughput fabrication of various biopolymer nanofibers with tailored conformational changes. We expect that fluid flow through nanopores will have a significant effect on the final conformation of the FN molecules as it controls the magnitude the elongational or extensional forces and shear stresses during the process of extrusion. However, given that our primary objective here is to present a proof of concept, which would be inexpensive but


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effective, we have opted to exploit only the manual mode of operation. Further, it will be especially exciting to analyze in more detail how conformational changes in protein nanofibers are affected by other external factors such as pH of the medium or the presence of other FN-binding proteins like heparin or collagen.

Acknowledgements The authors would like to thank Christine Mollenhauer for her intensive help with the FN purification and Neda Aslankoohi for her support in setting up the extrusion setup. The research leading to these results has received funding by the Max Planck Society and the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement no. 294852. Part of this work was supported by the BMBF/MPG network MaxSynBio. J.P.S. is the Weston Visiting Professor at the Weizmann Institute of Science and is a member of the Heidelberg cluster of excellence CellNetworks.

Notes The authors declare no competing financial interest.

References

1. Carlisle, C. R.; Coulais, C.; Guthold, M. Acta Biomater 2010, 6, 2997-3003. 2. Rho, K. S.; Jeong, L.; Lee, G.; Seo, B. M.; Park, Y. J.; Hong, S. D.; Roh, S.; Cho, J. J.; Park, W. H.; Min, B. M. Biomaterials 2006, 27, 1452-1461. 3. Matthews, J. A.; Wnek, G. E.; Simpson, D. G.; Bowlin, G. L. Biomacromolecules 2002, 3, 232-238. 4. Feinberg, A. W.; Parker, K. K. Nano Lett 2010, 10, 2184-2191. 5. Kubow, K. E.; Vukmirovic, R.; Zhe, L.; Klotzsch, E.; Smith, M. L.; Gourdon, D.; Luna, S.; Vogel, V. Nat Commun in press. 6. Little, W. C.; Smith, M. L.; Ebneter, U.; Vogel, V. Matrix Biol 2008, 27, 451461. 7. Mao, Y.; Schwarzbauer, J. E. Matrix Biol 2005, 24, 389-399. 8. Singh, P.; Carraher, C.; Schwarzbauer, J. E. Annu Rev Cell Dev Bi 2010, 26, 397-419. 9. Li, B. J.; Moshfegh, C.; Lin, Z.; Albuschies, J.; Vogel, V. Sci Rep-Uk 2013, 3, 18. 10. Vogel, V.; Sheetz, M. P. Curr Opin Cell Biol 2009, 21, 38-46. 11. Ahmed, Z.; Brown, R. A. Cell Motil Cytoskel 1999, 42, 331-343. 12. Baneyx, G.; Vogel, V. P Natl Acad Sci USA 1999, 96, 12518-12523. 13. Brown, R. A.; Blunn, G. W.; Ejim, O. S. Biomaterials 1994, 15, 457-464. 14. Cantini, M.; Rico, P.; Salmerón-Sánchez, M., Fibronectin Fibrillogenesis at the Cell–Material Interface. In Biomimetic Approaches for Biomaterials Development, Wiley-VCH Verlag GmbH & Co. KGaA: 2012; pp 189-212. 15. Chabria, M.; Hertig, S.; Smith, M. L.; Vogel, V. Nat Commun 2010, 1, 1-9.


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16. Deravi, L. F.; Su, T.; Paten, J. A.; Ruberti, J. W.; Bertoldi, K.; Parker, K. K. Nano Lett 2012, 12, 5587-5592. 17. Ejim, O. S.; Blunn, G. W.; Brown, R. A. Biomaterials 1993, 14, 743-748. 18. Kaiser, P.; Spatz, J. P. Soft Matter 2010, 6, 113-119. 19. Klotzsch, E.; Smith, M. L.; Kubow, K. E.; Muntwyler, S.; Little, W. C.; Beyeler, F.; Gourdon, D.; Nelson, B. J.; Vogel, V. P Natl Acad Sci USA 2009, 106, 1826718272. 20. Little, W. C.; Schwartlander, R.; Smith, M. L.; Gourdon, D.; Vogel, V. Nano Lett 2009, 9, 4158-4167. 21. Marco, C.; Cristina, G.-G.; Virginia, L.-H.; Manuel, S.-S., Material-Driven Fibronectin Fibrillogenesis. In Proteins at Interfaces III State of the Art, American Chemical Society: 2012; Vol. 1120, pp 471-496. 22. Nelea, V.; Kaartinen, M. T. J Struct Biol 2010, 170, 50-59. 23. Rico, P.; Hernandez, J. C. R.; Moratal, D.; Altankov, G.; Pradas, M. M.; Salmeron-Sanchez, M. Tissue Eng Pt A 2009, 15, 3271-3281. 24. Ulmer, J.; Geiger, B.; Spatz, J. P. Soft Matter 2008, 4, 1998-2007. 25. WojciakStothard, B.; Denyer, M.; Mishra, M.; Brown, R. A. In Vitro Cell DevAn 1997, 33, 110-117. 26. Nguyen, H. T. T.; Huynh, K. C.; Scharf, R. E.; Stoldt, V. R. Biol Chem 2013, 394, 1495-1503. 27. Liao, S.; Ramakrishna, S.; Ramalingam, M. J Biomater Tiss Eng 2011, 1, 111-128. 28. Khadka, D. B.; Haynie, D. T. Nanomed-Nanotechnol 2012, 8, 1242-1262. 29. Palchesko, R.; Sun, Y.; Zhang, L.; Szymanski, J.; Jallerat, Q.; Feinberg, A., Nanofiber Biomaterials. In Springer Handbook of Nanomaterials, Vajtai, R., Ed. Springer Berlin Heidelberg: 2013; pp 977-1010. 30. Scheibel, T. Curr Opin Biotech 2005, 16, 427-433. 31. Barnes, C. P.; Sell, S. A.; Boland, E. D.; Simpson, D. G.; Bowlin, G. L. Adv Drug Deliver Rev 2007, 59, 1413-1433. 32. Pham, Q. P.; Sharma, U.; Mikos, A. G. Tissue Eng 2006, 12, 1197-1211. 33. Hauser, C. A. E.; Zhang, S. G. Chem Soc Rev 2010, 39, 2780-2790. 34. Liu, X. H.; Ma, P. X. Biomaterials 2009, 30, 4094-4103. 35. Teo, W. E.; Inai, R.; Ramakrishna, S. Sci Technol Adv Mat 2011, 12, 1-19. 36. Kumbar, S. G.; James, R.; Nukavarapu, S. P.; Laurencin, C. T. Biomed Mater 2008, 3, 1-15. 37. Yoo, H. S.; Kim, T. G.; Park, T. G. Adv Drug Deliver Rev 2009, 61, 10331042. 38. Meinel, A. J.; Kubow, K. E.; Klotzsch, E.; Garcia-Fuentes, M.; Smith, M. L.; Vogel, V.; Merkle, H. P.; Meinel, L. Biomaterials 2009, 30, 3058-3067. 39. Mitsi, M.; Handschin, S.; Gerber, I.; Schwartlander, R.; Klotzsch, E.; Wepf, R.; Vogel, V. Biomaterials 2015, 36, 66-79. 40. Baneyx, G.; Baugh, L.; Vogel, V. Proc Natl Acad Sci U S A 2001, 98, 1446414468. 41. Smith, M. L.; Gourdon, D.; Little, W. C.; Kubow, K. E.; Eguiluz, R. A.; LunaMorris, S.; Vogel, V. PLoS biology 2007, 5, e268. 42. Raoufi, M.; Tranchida, D.; Schonherr, H. Langmuir 2012, 28, 1009110096. 43. Raoufi, M.; Schonherr, H. Rsc Adv 2013, 3, 13429-13436.


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44. Brüggemann, D.; Michael, K. E.; Wolfrum, B.; Offenhäusser, A. Int J Nano Biomat 2012, 4, 108-127. 45. Weber, D.; Mourzina, Y.; Brüggemann, D.; Offenhäusser, A. J Nanosci Nanotechnol 2011, 11, 1293-1296. 46. Bradshaw, M. J.; Cheung, M. C.; Ehrlich, D. J.; Smith, M. L. Plos Comput Biol 2012, 8, e1002845. 47. Baugh, L.; Vogel, V. J Biomed Mater Res A 2004, 69A, 525-534. 48. Rodriguez-Larrea, D.; Bayley, H. Nat Nanotechnol 2013, 8, 288-295. 49. Oukhaled, A.; Cressiot, B.; Bacri, L.; Pastoriza-Gallego, M.; Betton, J. M.; Bourhis, E.; Jede, R.; Gierak, J.; Auvray, L.; Pelta, J. Acs Nano 2011, 5, 3628-3638. 50. Cressiot, B.; Oukhaled, A.; Bacri, L.; Pelta, J. BioNanoSci. 2014, 4, 111-118. 51. Li, J. L.; Fologea, D.; Rollings, R.; Ledden, B. Protein Peptide Lett 2014, 21, 256-265. 52. Talaga, D. S.; Li, J. J Am Chem Soc 2009, 131, 9287-9297. 53. Benecky, M. J.; Kolvenbach, C. G.; Wine, R. W.; Diorio, J. P.; Mosesson, M. W. Biochemistry-Us 1990, 29, 3082-3091. 54. Martinez-Haya, B.; Gordillo, M. C. Macromol Theor Simul 2005, 14, 421427. 55. Aden, J.; Wittung-Stafshede, P. Biochemistry-Us 2014, 53, 2271-2277. 56. Dhar, A.; Samiotakis, A.; Ebbinghaus, S.; Nienhaus, L.; Homouz, D.; Gruebele, M.; Cheung, M. S. Proc Natl Acad Sci U S A 2010, 107, 17586-17591. 57. van den Berg, B.; Ellis, R. J.; Dobson, C. M. Embo J 1999, 18, 6927-6933. 58. Co, N. T.; Hu, C. K.; Li, M. S. The Journal of chemical physics 2013, 138, 185101. 59. Shin, J.; Cherstvy, A. G.; Metzler, R. Soft Matter 2015, 11, 472-488. 60. Zhou, H. X.; Rivas, G.; Minton, A. P. Annual review of biophysics 2008, 37, 375-397. 61. Zeiger, A. S.; Loe, F. C.; Li, R.; Raghunath, M.; Van Vliet, K. J. PloS one 2012, 7, e37904. 62. Peleg, O.; Savin, T.; Kolmakov, G. V.; Salib, I. G.; Balazs, A. C.; Kroger, M.; Vogel, V. Biophys J 2012, 103, 1909-1918. 63. Brockwell, D. J. Curr Nanosci 2007, 3, 3-15. 64. Lai, C. S.; Wolff, C. E.; Novello, D.; Griffone, L.; Cuniberti, C.; Molina, F.; Rocco, M. J Mol Biol 1993, 230, 625-640.


16

AAO

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Nano Letters

AAO

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1.0

0.5


Substrate

0

A

A

Feed solution Feed

Nano Letters

Page 18 of 22

solution

AAO

3 membrane 4 5 Substrate 6 Substrate 7 8 CC B910B DDD 11 12 13 14 15 16 17 ACS Paragon Plus Environment 18 19200 nm 1 μm 200 nm 1 μm Substrate 20

AAO

1 Mounted AAO Mounted AAO 2membrane

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A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

5 µm

C

500 nm

Nano Letters

B

500 nm

D

ACS Paragon Plus Environment 2 µm

A dAAO = 20 nm C = 1000 µg/ml

C

0.5

F

E

0 1.0

0.5

ACS Paragon Plus Environment 0

FRET Index

dAAO = 70 nm C = 500 µg/ml

D

1.0 Page 20 of 22

FRET Index

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

B

Nano Letters

A

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B

Diameter

Concentration

(Concentration: 1000 μg/ml)

(Pore diameter: 20 nm) 1000 μg/ml

Normalized number

200 nm

Normalized number

70 nm

500 μg/ml

20 nm

0.1

0.2

0.3

0.4

0.5

0.6

0.7

250 μg/ml

0.8

FRET index

100mg/ml μg/ml 0.10

0.1

C

0.2

0.3

0.4

0.5

FRET index FRET index

0.5

0.6

0.7

0.8

Free FN in PBS

20 nm 70 nm 200 nm

0.4

FRET index

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

0.3 Free FN with 1 M GdnHCl

0.2

Free FN with 4 M GdnHCl

0

200

400

600

Concentration (μg/ml ) ACS Paragon Plus Environment

800

1000

n

n

Nanopore Diameters Tune Strain in Extruded ... - ACS Publications

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