The Nanoscale Geometrical Maturation of Focal Adhesions Controls

Denmark. ⊥ Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands. Nano Lett. , 2014, 14 (7), pp 3945–3952. DO...
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The Nanoscale Geometrical Maturation of Focal Adhesions Controls Stem Cell Differentiation and Mechanotransduction Julien E. Gautrot,*,†,‡ Jenny Malmström,§ Maria Sundh,∥ Coert Margadant,⊥ Arnoud Sonnenberg,⊥ and Duncan S. Sutherland*,∥ †

Institute of Bioengineering and ‡School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom § School of Chemical Sciences, University of Auckland, Auckland, New Zealand ∥ Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark ⊥ Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands S Supporting Information *

ABSTRACT: We show that the nanoscale adhesion geometry controls the spreading and differentiation of epidermal stem cells. We find that cells respond to such hard nanopatterns similarly to their behavior on soft hydrogels. Cellular responses were seen to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction rather than impaired recruitment of proteins involved in focal adhesion formation. KEYWORDS: Keratinocyte, differentiation, nanoscale, mechanotransduction, vinculin, focal adhesion

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well-defined compositional architecture of FAs in the zdirection.9 Different integrins such as α5β1 and αvβ3 play different roles in matrix binding, cell signaling, and mechanosensing10 and display different nanoscale behavior within FAs.11 Adapter proteins such as talin, vinculin, and αactinin subsequently play a key role in FA formation and directly link ECM bound integrins to the cytoskeleton.12−14 Hence the molecular architecture of adhesion sites plays an important role in the formation of FAs, their dynamics, and their ability to generate and transduce forces. However, the impact of nanoscale architectural properties of the matrix on this molecular machinery and the mechanisms via which these biophysical cues dictate stem cell fate remain poorly understood. We recently found that epidermal stem cell fate was controlled by nanoscale mechanical properties of the matrix, rather than bulk properties.15 Given the importance of matrix adhesion,16 cell spreading and cell shape17 on the fate decision of epidermal stem cells, we made the hypothesis that other nanoscale properties of the matrix could play a similar role in regulating cell behavior and potentially allow very rigid matrices to mimic the impact of soft hydrogels on cell spreading and differentiation. In this report, we investigate the impact of nanoscale patterning of biointerfaces on epidermal stem cell differentiation and the role of the geometrical, compositional (more generally, biochemical), and mechanical maturation of nascent adhesions into FAs on cell spreading and fate decision.

he stem cell niche is defined by an intricate combination of biochemical and physical cues.1 Fate decision is tightly regulated by this microenvironment and cell adhesion to extracellular matrix (ECM) often plays an important role in this process.2 The study of such interactions is essential for our understanding of stem cell fate decision and tissue homeostasis in physiological as well pathological scenarios and for the design of biomaterials and engineered platforms for regenerative medicine applications. In this respect, several recent reports have highlighted the importance of nanoscale physicochemical properties of the ECM in dictating a wide range of behaviors such as spreading, motility, polarity, proliferation, differentiation, and apoptosis.3−5 How such nanoscale cues are transduced into molecular signaling events able to control phenomena such as cell spreading and stem cell fate decision is not fully understood. Focal adhesions (FAs) are micrometer-size complexes of proteins anchoring the cell cytoskeleton to the ECM via integrins and are considered as important transducers of physical cues.6,7 FAs arise from integrin clusters and nascent adhesions that mature in size and composition as they strengthen and ultimately allow the assembly of the f-actin cytoskeleton and the generation of tension, resulting in cell spreading. However, how this process is controlled at the molecular level and regulated by nanoscale physical properties of the ECM is not clearly understood. The nanoscale architecture of adhesion sites plays an important role for the formation of FAs and their dynamics: reports suggest the existence of a critical distance between integrin binding sites of ca. 70 nm and clustering of at least 4 ligand-occupied integrin heterodimers is required to sustain cell spreading.3−8 Super-resolution microscopy demonstrated the © XXXX American Chemical Society

Received: April 3, 2014 Revised: May 19, 2014

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Figure 1. Nanoscale geometry of adhesions controls epidermal stem cell spreading and differentiation. (A) Schematic representation of cells spreading on nanopatterns. Average cell densities (B), cell areas (C), and percentage of involucrin-positive cells (D) measured 24 h after keratinocyte seeding on nanopatterned substrates. Error bars are s.e.m., n ≥ 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Au and 10%Fn are homogeneous gold substrates coated with fibronectin and fibronectin/BSA 1/10 solutions (10 μg/mL total protein concentration), respectively; 3000, 600, 300, and 100 are the corresponding sizes (in nm) of the circular gold regions (in a silicon oxide background) of nanopatterned substrates coated with fibronectin (10 μg/mL); SiO2 correspond to homogeneous silicon oxide substrates coated with protein resistant PLL−PEG prior to incubation with fibronectin (10 μg/mL). (E) Immunofluorescence microscopy images of keratinocytes spreading and differentiating on nanopatterns (first three left columns, factin, and involucrin) and single cell images showing myosin IIa association to the actin cytoskeleton and vinculin sequestration to adhesion sites (right columns; the far-right column shows zooms of the regions of interest delimited by the dashed boxes).

differentiate was examined by immunofluorescence microscopy. We found a progressive restriction of cell spreading when comparing substrates with decreasing pattern sizes to homogeneous substrates (Figure 1C,E), while cell densities remained relatively high on all substrates (Figure 1B). This result is in good agreement with previous observations made on engineered substrates,18,20,21 although no area threshold was observed. This trend correlated with a gradual increase in keratinocyte commitment to terminal differentiation, as evidenced by changes in expression of the cornified envelope

To investigate the role of nanoscale geometrical maturation on keratinocyte spreading and fate decision, we generated nanopatterned substrates via colloidal lithography18 presenting circular gold regions with sizes ranging from 100 nm to 3 μm, coated with fibronectin and surrounded by protein- and cellresistant polymer brushes (see Figure 1A). Cells that spread on these substrates make adhesions selectively with the nanoscale fibronectin patches, hence enabling the control of the adhesion size.18,19 Human primary keratinocytes were seeded on the resulting substrates and their spreading and commitment to B

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proteins such as talin.28 These small protein complexes further recruit other proteins such as vinculin and paxillin at later stages, a phenomenon that correlates with the development of the f-actin cytoskeleton.6 Therefore, we explored the recruitment of these different proteins during the geometrical maturation of nascent adhesions to FAs, in cells seeded on nanopatterns of varying sizes, via immunofluorimetry (Figure 2A, Supporting Information Figures S2 and S3A). We found that the basal protein densities (intensity per cell area) were similar or slightly increasing in cells on the smallest nanopatterns (with factors ranging from 1.15 to 1.4, Figure 2A). However, due to the decrease in cell area, the total level of

precursor involucrin (Figure 1D,E). Differentiation levels remained lower than those observed for keratinocytes spreading on soft matrices (polyacrylamide hydrogels with Youngs’ moduli below 2 kPa),15 which may result from the stronger suppression of cell spreading on such compliant substrates. In order to test whether changes in cell spreading and differentiation were due to variations in the total amounts of extracellular matrix available for cell interactions, we also seeded keratinocytes on homogeneous substrates coated with 10% fibronectin (mixed with BSA). As for micropatterned substrates,17 no major change in cell spreading or increase in involucrin expression was observed even at 10% of the maximum ECM density (Figure 1B−D), confirming that above a certain threshold geometrical cues rather than total available ECM controls cell spreading and behavior. Finally, in order to ascertain if the size of adhesions formed was indeed controlled at the nanoscale, we probed the basal localization of the FA molecule vinculin via immuno-fluorescence microscopy (Figure 1E). We found an excellent correlation between the size of the vinculin patches and the size of the underlying patterns. Hence nanoscale confinement of adhesion sites and restriction of the normal geometrical maturation of FAs resulted in impaired cell spreading and increased commitment to cell differentiation, mimicking the response of cells to soft hydrogels. To explore the mechanism responsible to control such nanoscale-induced cell commitment, we tested the role of the mitogen-activated protein kinase (MAPK) pathway on involucrin expression. Focal adhesion formation by keratinocytes activates ERK phosphorylation, inhibiting JNK activity22,23 and resulting in decreased AP-1 transcriptional activity,24 an important family of transcription factors controlling the expression of epidermal terminal differentiation markers.25 We previously found that this pathway is implicated in the sensing of substrate nanoscale mechanics by keratinocytes.15 Cells seeded on homogeneous substrates and 100 nm patterns were incubated for 2 h and then treated with inhibitors of the MAPK kinase (MEK, PD0325901), the Jun Nterminal kinase (JNK, SP600125), or the transcription factor AP-1 (Tanshinone IIA) for a further 22 h (Supporting Information Figure S1). We found a significant reduction in keratinocyte differentiation on 100 nm nanopatterns when cells were treated with the JNK and AP-1 inhibitors (compared to carrier), suggesting that, similarly to the induction of keratinocyte differentiation on soft acrylamide hydrogels, the MAPK pathway, and associated JNK and AP-1 activation, control keratinocyte terminal differentiation.15 Similarly to suspension-induced differentiation (an assay in which cells are cultured in suspension), cells did not respond to treatment with the MEK inhibitor,26 perhaps highlighting the dual role that MEK can play in mediating ERK and PKC signaling pathways.23,27 Hence the transduction of different nanoscale physical signals (matrix mechanics and adhesion geometry) controlling keratinocyte differentiation is regulated by similar pathways and correlates with impaired cell spreading. These results suggest that modulation of FA geometry with ECM nanopatterns mimics soft matrices and provides a novel tool for controlling stem cell fate decision, independently of matrix stiffness. We next studied the recruitment of key focal adhesion proteins at the basal cell membrane. Focal adhesions are initiated as nascent adhesions that require the activation and clustering of several integrins (4−5)8 and recruitment of

Figure 2. Biochemical maturation of adhesion sites is decoupled from their geometrical maturation. (A) Total FA marker and (B) phosphorylation of adhesion markers basal density (intensity per cell area) measured for single cells spreading on nanopatterned substrates. Relative intensities are measured against cells adhering to homogeneous gold substrates. Error bars are s.e.m., n ≥ 3; at least 15 cells for each replicate for each condition; **, P < 0.01; see Supporting Information Tables S9 and S11 for statistical analysis. (C) Corresponding immunofluorescence images for p-FAK and p-cortactin (see Supporting Information Figure S2 for other examples) and laminin-332 deposition on nanopatterned substrates (green, laminin332; blue, DAPI; red, cell contour). Total cytoskeleton-associated marker (myosin IIa and α-actinin) basal intensity (D, intensity per cell) and density (E, intensity per cell area) measured for single cells spreading on nanopatterned substrates. Relative intensities are measured against cells adhering to homogeneous gold substrates. See Supporting Information Figure S9 for corresponding images. C

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proteins recruited at the basal membrane per cell was decreasing as a function of nanopattern size. These results indicate that protein recruitment is not impaired at the basal cell membrane of cells spreading on nanopatterns. Hence, the geometrical maturation of adhesion sites is decoupled from protein recruitment. The exception to this trend was vinculin, for which we measured a slight decrease in basal protein density. This suggests a different mechanism and role for the recruitment pattern of this protein during the establishment of large stable FAs, which is in agreement with its role in mechanotransduction.12,29 FAs act as mechanotransduction sites able to convert mechanical and physical cues into biochemical output and to alter cell behavior.7 Phosphorylation of proteins such as FAK and cortactin at adhesion sites has been implicated in the sensing of the physical properties of the microenvironment and in keratinocyte differentiation.30,31 To explore whether cell response to ECM nanoscale geometry was mediated by changes in protein phosphorylation, we measured protein recruitment and phosphorylation at the basal cell membrane, focusing on FAK, cortactin. and pan-phosphorylation of tyrosines (Figure 2B,C and Supporting Information Figures S2 and S3B). We found again that the basal density of phosphorylated proteins was not impaired on nanopatterns and even increased slightly (with factors above 1.5 for 100 nm patterns). This indicates that protein phosphorylation is regulated by matrix adhesion independently of the geometrical maturation of FAs (at least partially). Aside from protein phosphorylation, matrix adhesions also play an important role in the assembly of new matrix from secreted ECM proteins or proteins deposited from the surrounding medium.32 When spreading and moving along a substrate, keratinocytes leave trails of laminin-332 behind them.33 We found that this too was not impaired by nanoscale restriction of FA geometry and that cells deposited patches of laminin-332 that matched the size of the underlying nanopattern (Figure 2C and Supporting Information Figure S4). The 100 nm patterns were the only substrates for which these laminin-332 deposits did not form trails behind cells, but were rather localized underneath cells, implying that although matrix deposition is not impaired at this scale, cell motility is restricted. In order to gain further insight into the relationship between adhesions geometrical maturation and protein recruitment and phosphorylation, we developed a FA analysis protocol allowing the quantification of the frequency and intensity of individual adhesions as a function of their size (Figure 3 and Supporting Information Figures S5−S7 and methods). Consistent with the ability of nanopatterns to constrain adhesion size, we found a good correlation between the dimension of the fibronectin patches and the frequency distributions of adhesion sizes (Supporting Information Figure S6−7). The intensity distributions quantify the relationship between protein recruitment at adhesion sites and FA geometrical maturation; a strong coupling between those two phenomena will result in a steep increase in intensity as a function of adhesion size, whereas a lack of coupling will result in no or very moderate increase. On nonpatterned and large nanopatterned (3000 nm) substrates, talin and vinculin distributions displayed steady increases in intensity as a function of adhesion size (Figure 3 and Supporting Information Figures S6 and S7). A similar distribution was found for phosphorylated FAK and panphosphorylation of tyrosine residues, however, the activated form of β1-integrins34 showed a shallower profile, which is

Figure 3. Analysis of protein sequestration at single adhesion sites. (A) Distributions of mean intensities of talin positive objects. Relative intensities are measured against the intensity of the smallest object size measured for homogeneous gold substrates. Error bars are s.e.m., n = 3, n ≥ 10 cells for each experiment; n.s., nonsignificant; *, P < 0.05; **, P < 0.01. (B) Comparison of the average intensity of objects in the 0.30−0.43 μm2 and 2.7−3.8 μm2 range for adhesion-associated proteins (activated β1, talin, vinculin, p-FAK, and p-tyrosine) in cells spreading on nanopatterned substrates.

consistent with its early recruitment at adhesion sites. On smaller nanopatterns (600 nm), the intensity distributions of talin, vinculin, phosphorylated FAK (p-FAK), and tyrosines (pTyr) had shallow profiles. The abundance of these proteins at small adhesion sites (in the range of the underlying nanopattern size, that is, 0.3 μm2) did not decrease to the same extent as for homogeneous substrates or large nanopatterns. Hence the intensity of these distributions in the range of 0.3−0.43 μm2 was higher for the 600 nm nanopatterns than for the larger or nonpatterned substrates (Figure 3). This effect was such that the protein densities in this range were comparable to those of larger adhesions (2.7−3.8 μm2) on nonpatterned substrates for talin (P = 0.72), p-FAK (P = 0.17), and p-Tyr (P = 0.10). In contrast, vinculin recruitment to 0.3−0.43 μm2 adhesions on 600 nm nanopatterns remained markedly lower than for 2.7− 3.8 μm2 adhesions on nonpatterned substrates (P = 0.045), suggesting a stronger coupling between FA geometrical maturation and vinculin sequestration. Such coupling reflects the force-dependent sequestration of vinculin to talin at focal adhesions.12,13,29 Interestingly, protein densities were higher for 3000 nm nanopatterns compared to nonpatterned substrates, suggesting that constrains on the localization of adhesion sites lead to protein enrichment, perhaps via changes in adhesion D

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fiber stabilization rather than actin recruitment was impaired on nanopatterns. Similarly, we found that myosin IIa, which gives rise to f-actin contractility,39 was also recruited at the basal membrane (Supporting Information Figure S9D,F,G), although not forming dense mats as observed for nonpatterned and 3000 nm patterns. When treated with the myosin inhibitor blebbistatin, marked changes in cell shape and increased spreading were observed, even down to 300 nm nanopatterns, compared to nontreated cells on identical substrates (Supporting Information Figure S9C). Finally, α-actinin, an f-actin crosslinking protein forming dimers connecting actin bundles and competing with talin for integrin binding sites,14 was heavily recruited (factor of 1.5 ± 0.3 to 1.7 ± 0.4) to the basal membrane of cells spreading on the smallest nanopatterns (Supporting Information Figure S9E−G). This indicates a role of α-actinin in nanoscale sensing, perhaps owing to its involvement in myosin-independent nascent adhesion assembly.40 Together, these results show that the contractility resulting from the f-actin and myosin IIa assembly at nascent adhesion sites cannot be sustained by geometrically immature adhesions. In order to examine the role of vinculin dynamics in mediating the nanoscale geometrical sensing of adhesions, we carried out fluorescence recovery after photobleaching (FRAP) experiments. Keratinocytes were transfected with vinculinvenus and the rate of recovery of this tagged protein was quantified for cells spreading on homogeneous as well as nanopatterned substrates. The rate of recovery of vinculin measured for cells spreading on nonpatterned substrates (0.048 ± 0.003 s−1, Figure 4, Supporting Information Supplementary Videos 1−3) was in good agreement with that measured in previous studies (0.025 s−1)41 and was found to increase 2.5 fold when cells were treated with blebbistatin, which is in line with previous reports (2.9 fold).41 Although the vinculin recovery rate for cells spreading on 3000 nm substrates (0.053 ± 0.003 s−1) was close to that measured for homogeneous substrates, we observed a 2.2 fold increase when adhesions were restricted to 600 nm fibronectin patches, mirroring the sizedependent dynamics observed for FAK and paxillin.42 In order to explore how such reduced stability correlates with the mediation of tension, we used the recently developed vinculin tension sensor (VinTS).43 Förster resonance energy transfer (FRET) microscopy was carried out with cells expressing the vinculin tension sensor and spreading on homogeneous as well as nanopatterned substrates. Similarly to the relationship reported between adhesion size and vinculin-mediated tension during the FA assembly,43 we found that tension decreased with increasing size of adhesions for cells spreading on homogeneous and 3000 nm patterns (Supporting Information Figure S10) and the residual tension mediated by vinculin on fully mature FAs was not detectable with the tension sensor (compared to the tail-less mutant VinTL control unable to mediate tension43). For cells spreading on 600 nm patterns, FRET indexes were similar to those measured for cells treated with blebbistatin or VinTL. Hence, the nanoscale frustration of adhesions results in destabilization of vinculin and its inability to mediate tension in developing focal adhesions, ultimately preventing the maturation of a stable f-actin cytoskeleton. To further probe the relationship between nanoscale geometrical maturation and mechanical coupling of adhesion sites, we studied the influence of specific integrin heterodimer expression on nanoscale restriction of cell spreading. Previous studies found that β1 and β3 integrins regulate distinct

dynamics and turnover. Hence, our results show that nanoscale restriction of adhesion size does not prevent further recruitment of FA-associated proteins, except in the case of vinculin. We propose that the role of vinculin in the regulation of tension at growing FAs (by providing a mechanical link between the integrin tail and the actin cytoskeleton13) is decoupled from the recruitment and biochemical maturation of other proteins at adhesion sites. Several types of integrins contribute to keratinocyte anchoring to their matrix and cell spreading.16 While the collagen and laminin receptors α2β1 and α3β1 are involved in FA formation, the laminin receptor α6β4 regulates the formation of hemidesmosomes, large (several micrometers) complexes of proteins anchoring the ECM to the keratin intermediate filament network.35 Importantly, these adhesions, which are not directly linked to the f-actin cytoskeleton, are not thought to contribute to mechanical tension. Considering the importance of these adhesive structures to keratinocyte spreading, motility, and the maintenance of the epithelial sheet integrity in the interfollicular epidermis,36 we explored their response to nanoscale restriction (Supporting Information Figure S8). In addition, comparison of the protein recruitment of FA markers to that of proteins involved in the formation of these non-load-bearing adhesions should highlight the role of mechanotransduction mechanisms in nanoscale sensing. Similarly to FA proteins, we found that α6 integrin and the hemidesmosomal protein plectin were recruited to the basal membrane of cells spreading on small nanopatterns (basal densities of 1.2 ± 0.2 and 1.27 ± 0.09, respectively, on 100 nm patterns). In addition, the α6 distribution displayed a shallow size-dependent profile, as for activated β1 integrins, and recruitment was not prevented on 600 nm patterns (Supporting Information Figure S8). Hence our results indicate that protein recruitment to non-load-bearing adhesions is not coupled to adhesion size and is not restricted by nanoscale geometrical constraints. Our focal adhesion analysis gives direct evidence of the decoupling of the geometrical maturation of FAs and their biochemical maturations; early as well as late adhesion proteins are recruited to nanoscale-constrained adhesions and protein phosphorylation and matrix assembly are not prevented. However, cell spreading is progressively impaired, suggesting a coupling between FA geometrical maturation, f-actin cytoskeleton assembly, and mechanical maturation. The results we obtained for vinculin are consistent with this hypothesis; it was shown that vinculin is recruited in a force dependent manner for large focal adhesions (>1 μm2)37 as well as nascent focal contacts.38 Vinculin recruitment is activated by tension generated along the talin rod, exposing cryptic vinculin binding sites,12 and its active form conformation is stabilized by tension.13 Hence, the reduced recruitment of vinculin on nanopatterns, which contrasts with the recruitment of other adapter proteins, suggests a reduced mechanical tension as a result of the frustration of FA geometrical maturation. We investigated further the assembly of the actin cytoskeleton, vinculin dynamics, and mechanical coupling in keratinocytes spreading on nanopatterned substrates. Whereas keratinocytes spreading on nonpatterned or 3000 nm patterns generated a well-defined actin cytoskeleton with multiple actin fibers forming mats connecting FAs, actin fibers were sparser on smaller patterns (