Quantitative Analyses of Dynamic Features of Fibroblasts on Different

Article pubs.acs.org/journal/abseba

Quantitative Analyses of Dynamic Features of Fibroblasts on Different Protein-Coated Compliant Substrates Yue Xu,† Jing Li,† Shuai Zhou,† Xuan Tang,‡ Yanli Zhang,‡ Feng Lin,§,⊥ Chunyang Xiong,*,§,⊥ and Chun Yang*,† †

Institute of Biomechanics and Medical Engineering, School of Aerospace Engineering, and ‡Cell Imaging Platform of Protein Research Technology Center, Tsinghua University, Beijing 10084, People’s Republic of China § State Key Laboratory for Turbulence and Complex System, and Department of Mechanics and Engineering Science, College of Engineering, and ⊥Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, People’s Republic of China S Supporting Information *

ABSTRACT: Cell response to substrate rigidity, closely related to extracellular matrix protein composition, requires actomyosin-generated contractility. By introducing coefficients describing cell spreading and traction dynamics, and a revised high-resolution traction force microscopy, we analyzed the static and dynamic features of fibroblasts on fibronectin- or collagen- coated stiff or soft substrates. Large cell spreading area and branchlike morphology were more favorable on fibronectin than collagen. Cell spreading on fibronectin-coated substrates was more sensitive to rigidity compared with collagen. Low concentration fibronectin-coated substrate induced more dynamic lamellipodia movement than other conditions. Interestingly, the static average cell traction on high concentration fibronectin-coated stiff and soft substrates showed no difference. However, the lamellipodium traction dynamics was sensitive to rigidity on fibronectin. Particularly, lamellipodia on fibronectin-coated soft substrate performed much higher local traction dynamics compared with other groups. Together, dynamics of cell adhesion and traction are regulated by extracellular matrix protein composition, coupled with substrate rigidity. KEYWORDS: high-resolution traction force microscopy, dynamic, substrate rigidity, ECM protein composition, fibroblast

■

traction.12 These studies imply that cell traction may be a critical factor for the coupling effect of ECM rigidity and protein composition on regulating cell behaviors. Benefitting from the development of micro- and nanotechnologies in recent years, independent groups have carried out multiscale research on the dynamic organizations of the FAs and cell contractions to get insight into the interactions between cells and ECM. Super-resolution microscopy has revealed the recruitment and rearward movements of β1integrins inside FAs.13 Our previous work based on atomic force microscopy has demonstrated that cells plated on soft substrates had a lower lifetime of integrin−ligand bond compared to those on stiff substrates.14 High-resolution traction force microscopy has suggested that fluctuations of force local maxima position within FAs mediate substrate rigidity sensing.15 These findings provide suggestive clues to mechanisms by which ECM controls cell traction and regulates cell behaviors.

INTRODUCTION Cells respond to their environment by sensing and transducing the environmental cues to guide various cell behaviors, including proliferation, differentiation, migration, and apoptosis.1−4 Such cues may be biochemical, like extracellular matrix (ECM) protein composition; or mechanical, like substrate rigidity. Several recent reports demonstrated that cell response to ECM rigidity is associated with the ECM protein composition. For example, mesenchymal stem cells plated on type I collagen (CL)-coated stiff elastic substrates undergo osteogenic differentiation, while those on fibronectin (FN)- and laminin-coated substrates, of the same rigidity, expressed lower level of osteogenic markers.5−8 Independent groups have shown that normal and nonmetastatic tumor cells respond to substrate rigidity on FN but not CL.9 These findings suggested that ECM protein composition is coupled with substrate rigidity in cell response to ECM. Cells forge connections to ECM via focal adhesion (FA) plaques, which consist of transmembrane integrins and a complex of cytoplasmic proteins linking ECM to the actin cytoskeleton.10 Integrin engagement to ECM via FAs and subsequent actomyosin-generated contractility are required for substrate rigidity sensing.11 Different ECM proteins, binding specific types of integrins, are also reported to affect cell © XXXX American Chemical Society

Special Issue: Multiscale Biological Materials and Systems: Integration of Experiment, Modeling, and Theory Received: September 29, 2016 Accepted: January 20, 2017 Published: January 20, 2017 A

DOI: 10.1021/acsbiomaterials.6b00598 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 1. ECM protein composition and substrate rigidity affect cell morphology and spreading area. (a) Schematic of the segmentation-based analysis method quantifying cell morphology. Cells were divided into 360 sectors with central angle of 1 degree. (b, c) Representative confocal images of EGFP-transfected NIH3T3 plated on (b) high- and (c) low-concentration FN- or CL-coated PAAm substrates for 2 h (scale bar: 10 μm). (d) Cell spreading area of NIH3T3 plated on the substrates under different ECM conditions for 2 h. (mean ± SD, n = 20 cells) *, p < 0.05; **, p < 0.001; ***, p < 0.0001; n.s., not significant.

compared with other groups, suggesting these cells undergoing an active bonds turnover. Together, our findings showed that ECM protein composition and substrate rigidity have sound impact on the dynamics of cell adhesion and traction magnitude, providing a new approach to the mechanism underlying the biomechanical and chemical coupling process in cell response to ECM.

Here, we sought to understand how ECM protein composition, coupled with substrate rigidity, regulates cell adhesion and traction magnitude dynamics. By plating NIH3T3 cells (a well-documented fibroblast cell line) on FN- or CLcoated stiff (90 kPa) or soft (12.3 kPa) substrates, we characterized cell morphologies and spreading area. FN-coated substrates promoted branchlike morphologies and large spreading area of the cells. Dynamic analysis of cell spreading showed that low concentration FN-coated substrate induced more dynamic lamellipodium movement than other ECM conditions. Despite these differences in cell spreading, the static average traction of the cells on high-concentration FN-coated stiff and soft substrate showed no difference. Other ECM conditions induced lower average traction on stiff substrate comparing with soft. Interestingly, the dynamic analysis on cell traction revealed that the lamellipodium traction dynamics was sensitive to rigidity on both high- and low-concentration FNcoated substrates; lamellipodia on FN-coated soft substrate performed a much higher local traction dynamics when

■

MATERIALS AND METHODS

Cell Culture and Transfection. NIH3T3 cells were maintained at 37 °C in DMEM (Corning) with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin at 5% CO2. For experiments, 1 × 106 cells were transfected with 5 μg of DNA encoding EGFP using a Neon Transfection System (Invitrogen), pulse voltage 1350 V, for 20 ms twice. Twenty-four hours after transfection, cells were replated on the polyacrylamide substrates described below. Revised High-Resolution Traction Force Microscopy. We modified a previously published protocol for high-resolution traction force microscopy.16 Briefly, 39 μm thick polyacrylamide (PAAm) substrates with variable Young’s Moduli were attached to a ϕ35 mm B


Article


Figure 2. Quantification of cell morphology. (a-b) Distribution of local radius measured from each sector of individual cells plated on the substrates coated with (a) high- and (b) low-concentration FN or CL for 2 h. Each color stands for one cell. Fraction of data points greater than 1.75 in each group were shown in the upper right corner. (c, d) Stacked distribution and Gaussian fits for local radius in the cells plated on the substrates coated with (c) high and (d) low concentration FN or CL. The standard deviation value σ of the fitted Gaussian distribution was shown in the legend box. (n = 20 cells). glass bottom dish (In-Vitro-Scientific). To avoid the sampling errors caused by the z-axis drift of the microscope, we linked 40 nm red fluorescent microspheres to the surface of the PAAm substrates for cell-induced substrate deformation visualizing,17 instead of embedding the microspheres inside the substrates. Benefitting from this modification, we could achieve a sampling resolution equivalent to a previous high-resolution protocol without a second type of fluorescent microspheres introduced therein, thus avoid the phototoxicity and time-consumption. Type I collagen or fibronectin were then crosslinked to the substrate surface at the concentration of 0.2 or 0.01 mg/ mL to facilitate cell adhesion.18 Subsequently, NIH3T3 cells expressing enhanced green fluorescent protein (EGFP) were plated onto the substrates at the density of 1 × 104/mL for 2 or 20 h. Images of the cells and microspheres on the substrates were captured by a laser scanning confocal microscope (LSCM). For time-lapse microscopy, cells were plated onto the substrates for 2 h, and images were captured at 1 min intervals for 60 min. Then, 500 mM NaOH were perfused into the dish with final concentration of 25 mM to detach the cells from the substrates, and images of microspheres on the undeformed substrates were captured immediately. Customgenerated Matlab programs were used for data processing.

Correlation-based particle tracking velocimetry (PTV) was utilized to quantify the displacement field of the microspheres on the substrates corresponding to the cell-induced substrate deformation. The displacement field was interpolated onto a regularized grid and the stress field was calculated by regularized-Fourier transform traction cytometry (reg-FTTC).16 The regularization parameter was set as 1 × 10−6 to achieve high resolution for the traction maps. More detailed experimental and computational process were described in the Supporting Information. Immunostaining. NIH3T3 were plated on ECM protein-coated PAAm substrates for 2 or 20 h before immunostaining. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeablized with 0.1% Triton X-100 for 5 min and blocked by 5% BSA for 1 h at room temperature. The cells were then incubated with primary antibody against FN (1:100, BD Biosciences) overnight at 4 °C and FITC-conjugated secondary antibody (1:100, Santa Cruz Biotechnology) for 2 h at room temperature. The cells were incubated with DAPI (Sigma-Aldrich) for 15 min before imaging. Statistical Analysis. Data are presented as means ± SD. Student’s t test was used to compare differences between two experimental C


Article


Figure 3. Time-lapse microscopy of cells on high concentration FN-coated substrates. (a, b) Analyses of one representative cell movie on (a) stiff and (b) soft substrates. Movie screenshot of EGFP-transfected NIH3T3 plated on the substrates (left) (scale bar 10 μm). Kymograph projection of the cell edge area marked by white line on the left. The longitudinal axis represents the outward direction perpendicular to the cell edge (bottom view, right). Time-averaged local radius (gray) and standard deviation (black) of each sector in the cells. Representative static (hollow arrow) and dynamic (solid arrow) lamellipodia are marked (top view, right). groups. P value in statistical graphs was described as follows: *, p < 0.05; **, p < 0.001; ***, p < 0.0001; n.s., not significant.

local radius, was recorded and normalized by the cell radius, an average of the local radius value in each cell. The normalized local radius were then plotted into a histogram; thus the loose and right-skewed distribution indicates branchlike morphology of the cells. Histogram analysis showed that local radius distributions of cells on FN-coated substrates skewed to the right, while the distribution of those on CL-coated substrates were almost symmetrical (Figure 2a, b). The stacked histogram, graphed by the local radius of all the cells under each ECM condition, showed a looser distribution on FN-coated substrates compared with CL-coated ones (Figure 2c, d). The cell response to ECM may change at longer time periods as adhesions maturing and matrix remodeling occur.19 Thus, we investigated morphologies of well-spread cells, plated on substrates for 20 h. The results showed that cells plated on CL-coated substrates also performed a right-skewed local radius distribution, analogous to that on FN-coated substrates. (Figure S1) In other words, the differences triggered by ECM condition weakened. Together, these results demonstrated that FN-coated substrates promoted branchlike cell morphology, whereas CL-

■

RESULTS Fibronectin-Coated Substrate Promotes Branchlike Morphology of Fibroblast during Early Cell Spreading. To understand the role of ECM protein composition and substrate rigidity in cell response to ECM, we first sought to investigate cell morphologies under different conditions. NIH3T3 cells expressing enhanced green fluorescent protein (EGFP) were plated on high (0.2 mg/mL) or low (0.01 mg/ mL) concentration FN- or CL- coated elastic polyacrylamide (PAAm) substrates of stiff or soft rigidity for 2 h and subjected to laser scanning confocal microscopy (LSCM). Microscopy showed that cells plated on FN-coated substrates had large spreading area and branchlike morphology, whereas CL-coated ones promoted small spreading area and relatively smooth cell edges (Figures 1b−d and Table S1). To quantify the cell morphology, we introduced a sectorsegmentation-based statistical method. With the cell centroid as the center, the cell was divided into 360 sectors with a central angle of 1° (Figure 1a). The radius of each sector, defined as D


Article


Figure 4. Time-lapse microscopy of cells on high concentration CL-coated substrates. (a, b) Analyses of one representative cell movie on (a) stiff and (b) soft substrates. Movie screenshot of EGFP-transfected NIH3T3 plated on the substrates (left) (scale bar: 10 μm) Kymograph projection of the cell edge area marked by white line on the left. The longitudinal axis represents the outward direction perpendicular to the cell edge (bottom view, right). Time-averaged local radius (gray) and standard deviation (black) of each sector in the cells. Representative static (hollow arrow) lamellipodia were marked (top view, right).

lamellipodia were more dynamic on FN compared to CL. (Figures 5 and 6, bottom view, right) Interestingly, cells on stiff and soft substrates had different dynamic patterns. Lamellipodia of cells on stiff substrates underwent a substantial rise or fall over time, whereas those on soft substrates underwent a “fluctuated” state (the lamellipodia edge move back and forth). High concentration ligand modification reduced the impact of substrate rigidity and ECM protein composition on cell spreading. (Figures 3 and 4) To quantify cell edge dynamics, we introduced two factors, the dynamic amplitude (δ) and the persistence coefficient (ζ). As the local radius at each time point (rti) in a cell had been measured, we calculated their differences between adjacent time points for each sector. We defined the time-averaged absolute value of the differences as the dynamic amplitude, i.e., 1 n δ = n ∑i = 2 |rti − rti−1|. n was the number of time points. For the persistence coefficient, we set the initial value of ζ to 0, then performed ζ = ζ + 1 upon the local radius increase, whereas we performed ζ = ζ − 1 upon the local radius decrease at each 1 n time point, i.e., ζ = n ∑i = 2 sgn(rti − rti−1). Thus, a large dynamic amplitude indicated a more dynamic lamellipodia,

coated ones promoted relatively smooth cell edge during early cell spreading. Spreading and Adhesion of Fibroblast Are Unstable on Low-Concentration FN-Coated Substrates. Cell morphology is affected by ECM protein composition, coupled with substrate rigidity. Such effects were comparatively strong during early stage of cell spreading, which was a highly dynamic process compared with the well-spread stage.19 We thus performed time-lapse microscopy to evaluate the dynamics of cell adhesion. A 60 min long movie (1 min image intervals) of an NIH3T3 cell expressing EGFP under different ECM conditions was captured by LSCM. (Figure 3−7 and Movies S1 and S2) With the time-averaged cell centroid as the center, the cell was divided into 360 sectors at each time point and the local radius were measured (Figure 1a). The time-averaged local radius and standard deviation of each sector were expanded into a Cartesian coordinate diagram (Figures 3−6, top view, right). On low-concentration ligand-coated substrates, the cells on FN possessed more protrusive (higher radius value) and dynamic (larger standard deviation) lamellipodia (indicated by solid arrows) comparing to the cells on CL, which had more stable lamellipodia (indicated by hollow arrows). Kymograph projections of the cell edge region also showed that E


Article


Figure 5. Time-lapse microscopy of cells on low concentration FN-coated substrates. (a, b) Analyses of one representative cell movie on (a) stiff and (b) soft substrates. Movie screenshot of EGFP-transfected NIH3T3 plated on the substrates (left). (Scale bar: 10 μm) Kymograph projection of the cell edge area marked by white line on the left. The longitudinal axis represents the outward direction perpendicular to the cell edge (bottom view, right). Time-averaged local radius (gray) and standard deviation (black) of each sector in the cells. Representative static (hollow arrow) and dynamic (solid arrow) lamellipodia were marked (top view, right).

i.e., a large variation in the lamellipodium length; a small persistence coefficient implied a “fluctuated” state described above. ECM protein composition had impact on cell spreading dynamics. On low concentration ligand-coated substrates, the values of δ on FN (7.2 on stiff substrate, 5.4 on soft substrate) are much higher than those on CL. However, high concentration ligand modification reduced the impact of ligand composition on the values of δ (about 4.0 under each condition) (Figure 7a). These results suggested that the lamellipodia on low concentration FN-coated substrates performed more dynamic movement than other groups. Ligand composition has impact on the response of cell spreading to substrate rigidity. Increasing CL concentration from 0.01 to 0.2 mg/mL reduced the difference of δ between stiff and soft from 36 to 7%, and diminished the difference of ζ; increasing FN concentration from 0.01 to 0.2 mg/mL reduced the difference of δ between stiff and soft from 25 to 18%, and ζ from 22 to 12%. Together, cell spreading on FN-coated substrates perform consistent rigidity sensitivity under various

ligand concentration, whereas the rigidity sensitivity vanish on CL-coated substrates at high ligand concentration (Figure 7 and Table S1) Fibroblast Varies in Average Traction under Different ECM Conditions during Early Cell Spreading. Because both morphological change and FA assembly are closely associated with cell traction, we next sought to determine the role of ECM protein composition and substrate rigidity in regulating cell traction by utilizing a revised high-resolution traction force microscopy (HR-TFM). NIH3T3 cells expressing EGFP were plated on FN- or CL-coated PAAm substrates of different rigidities. Red fluorescent beads (40 nm) were cross-linked to the surface of the substrates to visualize the cellinduced deformation under LSCM. The traction fields were reconstructed at 0.7 μm resolution with regularized-Fourier transform traction cytometry (Reg-FTTC). According to Nyquist sampling theorem, the reliable resolution was up to 1.4 μm, which met the requirements for studying traction in a single lamellipodium. F


Article


Figure 6. Time-lapse microscopy of cells on low-concentration CL-coated substrates. (a, b) Analyses of one representative cell movie on (a) stiff and (b) soft substrates. Movie screenshot of EGFP-transfected NIH3T3 plated on the substrates (left) (scale bar: 10 μm) Kymograph projection of the cell edge area marked by white line on the left. The longitudinal axis represents the outward direction perpendicular to the cell edge (bottom view, right). Time-averaged local radius (gray) and standard deviation (black) of each sector in the cells. Representative static (hollow arrow) lamellipodia were marked (top view, right).

Figure 7. Cell radius dynamic amplitude (left) and persistence coefficient (right) of NIH3T3 plated on the substrates under different ECM conditions (mean ± SD, n = 6 cells). *, p < 0.05; **, p < 0.001; ***, p < 0.0001; n.s., not significant.

3.18 on FN- and CL-coated substrates, respectively. (Figure 8b, c) For the well-spread cells, no significant difference of traction was observed in the cells under different ECM conditions. (Figure S2) On high concentration ligand-coated substrates, however, no difference in average traction of cells on FN-coated substrates

HR-TFM of early spreading cells showed that, on low concentration ligand-coated substrate, cells plated on FNcoated substrates applied larger average traction than those on CL-coated ones. (Figure 8b, c) Average traction of cells plated on soft substrates were larger than those on stiff substrates with same protein-coated, and the ratio of soft to stiff were 1.54 and G


Article


Figure 8. ECM protein composition and substrate rigidity affect cell traction. (a, b) Representative confocal images of EGFP-transfected NIH3T3 plated on the substrates coated with (a) high- and (b) low-concentration FN or CL for 2 h and corresponding maps of reconstructed traction stresses (scale bar: 10 μm). (c) Average traction of cells plated on the substrates under different ECM conditions (mean ± SD, n = 20 cells). *, p < 0.05; **, p < 0.001; ***, p < 0.0001; n.s., not significant.

conditions may be a key to understanding the mechanism of cell rigidity sensing. Sixty-minute-long movies of NIH3T3 cells expressing EGFP and cell-induced ECM deformation were captured by LSCM, and traction fields were reconstructed by reg-FTTC (Figures 9 and 10, Movie S3 and S4). We focused on measuring traction within protruding lamellipodia, a distinct adhesion-associated actin structure mediating cell interactions with ECM.20 To analyze traction magnitude within the lamellipodium, a binary region mask was created by intensity thresholding of the EGFP image at the region of a lamellipodium, and dilated by 2 μm to avoid the error raised by the limitation of resolution. Average traction magnitude of each lamellipodium at each time point (pti) was calculated and plotted, showing variation of lamellipodium traction over time. (Figure 9 and 10a−d, top view, right) To describe the dynamics of lamellipodium traction, we introduced the lamellipodium traction dynamic amplitude (Δ)

of different rigidity was seen, while average traction of cells plated on CL-soft substrates were larger than those on stiff ones. (Figure 8a, c) These results suggested that average cell traction are more sensitive to substrate rigidity on CL-coated substrates than on low concentration FN-coated ones in the early stage of cell spreading. ECM Condition Influence Lamellipodium Traction Magnitude Dynamics of Fibroblast. The dynamic analysis of cell spreading showed that cell shapes are more sensitive to rigidity on FN-coated than CL-coated substrate. Although cell spreading is regarded to be closely associated with cell adhesions and traction, we did not observe the difference in cell average traction on high concentration FN-coated stiff and soft substrate. Average traction or static traction is an analytical data that screened the dynamic processes, while shifting of cell traction local maxima position within focal adhesions had been reported on compliant substrate.15 We thus propose that investigating traction dynamics of cells under various ECM H


Article


Figure 9. Traction dynamic analysis of NIH3T3 on high concentration FN- or CL- coated substrates. (a−d) Analyses of one representative cell TFM movie under each condition. Movie screenshot (top view, left) of EGFP-transfected NIH3T3 plated on the substrates and corresponding reconstructed traction stresses (bottom view, left) (scale bar: 10 μm). Zoomed area illustrating one representative lamellipodium marked by yellow box on the left (scale bar: 5 μm) (bottom view, right). A representative local maxima point for Z analysis at each lamellipodium was marked by white asterisk. The top view on the right is the traction dynamics of the lamellipodia in the bottom view.

and local traction dynamic amplitude (Z). As the traction in a lamellipodium (pti) had been measured, we calculated their differences between adjacent time points. We defined the timeaveraged absolute value of the differences as the dynamic 1 n amplitude Δ, i.e., Δ = n ∑i = 2 |pt − pt |. Z was calculated i

groups on FN. However, interestingly, although the lamellipodium dynamics was much lower in the cells on FN-coated soft substrate, they performed a much higher Z when compared with other groups (Figure 11 and Table S1). These suggested that the cells on FN-coated soft substrate undergo an active bond turnover even if the total lamellipodium traction remained stable at high ligand concentration.

i−1

similarly. We first select the local maxima of the traction in each lamellipodium at the beginning of the observation. The traction at these specific points (qti) was measured and the average absolute differences between adjacent time points were 1 n calculated, i.e., Ζ = n ∑i = 2 |qt − qt |. Lamellipodium traction i

■

DISCUSSION

The present work demonstrated that the influence of ECM conditions weakened when cells well-spread on the substrates. The immunostaining of NIH3T3 on CL-coated substrates showed that cells deposit and reorganize FN after being plated for 20 h (Figure S3). The difference of ECM protein composition may have started to be concealed then. This result suggested that fibrillogenesis is a plausible reason for the faint fibroblast shape and traction response to ECM composition and substrate rigidity after 20 h. Cells plated on the soft substrates (12.3 kPa, close to the rigidity of connective tissue supporting fibroblast in vivo21) were found to apply larger traction than those on the stiff substrates (90 kPa, close to the rigidity of osseous tissue in

i−1

is the force forged by the whole lamellipodium, whereas local traction at specific points represents the force on molecule bonds in the vicinity. Thus, a large Δ indicated dynamic lamellipodia traction, and a large Z implied an active adherent bond turnover. On low concentration ligand-coated substrates, cells possessed larger Δ on FN than those on CL; Soft substrates induced a larger Z when compared with those on stiff substrates coated with same protein (Figure 11 and Table S1). On high concentration ligand-coated substrates, cells on CL demonstrated a much higher value of Δ when compared with I


Article


Figure 10. Traction dynamic analysis of NIH3T3 on low concentration FN- or CL- coated substrates. (a−d) Analyses of one representative cell TFM movie under each condition. Movie screenshot (top view, left) of EGFP-transfected NIH3T3 plated on the substrates and corresponding reconstructed traction stresses (bottom view, left) (scale bar: 10 μm). Zoomed area illustrating one representative lamellipodium marked by yellow box on the left (scale bar: 5 μm) (bottom view, right). A representative local maxima point for Z analysis at each lamellipodium was marked by white asterisk. The top view on right is the traction dynamics of the lamellipodia in the bottom view.

Figure 11. Lamellipodium traction dynamic amplitude (left) and local traction dynamic amplitude (right) of cells plated on the substrates under different ECM conditions. (mean ± SD, n > 20 lamellipodia, n = 12 local maxima points) *, p < 0.05; **, p < 0.001; ***, p < 0.0001; n.s., not significant.

vivo21). The appropriate stiffness of the former matrix may account for the higher cell traction of fibroblast on it. The present work showed that fibroblasts on low concentration FN-coated substrates applied larger traction than those on CL-coated ones, different from our previous studies on mesenchymal stem cells (MSC).22 This may be

attributed to the expressing level of different subunits of integrins in these cells. Fibroblasts express more α5-integrin (mainly associated with fibronectin) than α1- and α2- (mainly associated with type I collagen),23 whereas MSCs are the opposite.24 These suggest that specific pairing between integrin J



■

subunits and ligands contribute to the mechanism of ECM protein composition-mediated substrate rigidity sensing. Quantitative analyses have been contributing to illuminating the mechanism underlying various biological processes. As an approach to quantitative analysis, we have designed several coefficients to elucidate the static and dynamic features of the cells. Lamellipodia protrusion and retraction are regarded as a result of active actin bundles and traction reorganizing. However, the static analysis on average traction of the cells showed no difference between the cells on high concentration FN-coated stiff and soft substrate, despite the difference in cell spreading. The dynamic analysis on cell traction revealed that lamellipodia on FN-coated soft substrate performed a high local traction dynamic amplitude Z, while the lamellipodia traction dynamic amplitude Δ was rather stable when compared with those under other ECM conditions. Local traction represents the force on adherent molecule bonds in the vicinity. The stable Δ and the variation in Z suggested that the adherent bonds disassembled frequently and reassembled in other positions in the lamellipodia, keeping lamellipodia traction stable. The dynamic observation with HR-TFM may provide cues to uncover the intertwined biomechanical and biochemical process. FN-coated soft substrates induced active local traction variation in lamellipodia while CL-coated substrates brought stable ones. Since FA, anchorage of cell body and lamellipodium to ECM, contains a large number of integrinligand bonds, we propose that the lifetime of the bonds and the trafficking of integrins affect the stability of FAs and local traction variation. We have demonstrated in our previous study that cells plated on substrates of stiff and soft rigidity possessed dramatic differences in integrin-ligand bond lifetime (90s on stiff substrates and 2s on soft substrates),14 and the trafficking of β1 integrin contributed to stem cell differentiation on soft substrates;25 bonds of different integrin subunits and their ligands may also vary in lifetime and the trafficking pathways. Thus, studies on the dynamic properties of these bonds and the adherent molecule trafficking may shed light on understanding the cell response to ECM.

■

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00598. Extended methods and materials, Figures S1−S4, Table S1 (PDF) Movie S1, representative confocal movies of NIH3T3 cells expressing EGFP plated on high-concentration FNor CL-coated stiff or soft substrates (AVI) Movie S2, representative confocal movies of NIH3T3 cells expressing EGFP plated on low-concentration FNor CL-coated stiff or soft substrates (AVI) Movie S3, representative movies of lamellipodium traction map of NIH3T3 cells plated on highconcentration FN- or CL-coated stiff or soft substrates (AVI) Movie S4, representative movies of lamellipodium traction map of NIH3T3 cells plated on low concentration FN- or CL-coated stiff or soft substrates (AVI)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Chun Yang: 0000-0002-2562-2450 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This work was supported by the National Basic Research Program of China under Grant 2013CB933702 and the National Natural Science Foundation of China (NSFC) (31370939, 31400799 and 11472013). Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Zaicun Wang for technical assistance and helpful discussion.

CONCLUSIONS

Connections between cells and their ECM are essential for cell functions. Mechanical cues in cellular microenvironment have been brought to intensive attention recently. Mechanical environment used to be regarded as an independent external condition in regulating cell activities. However, chemical environmental factors, including ECM protein composition, have now been found to affect cellular mechanosensing. The single-fluorescence-channel high-resolution traction force microscopy that we introduced is a powerful approach to assay the biomechanical-biochemical coupled process. The sector-segmentation-based analysis and the coefficients (δ, ζ, Δ, and Z) defined here are helpful for featuring the static and dynamic properties of the morphology and traction of fibroblasts. The dynamic analysis methods revealed subtle traction variation hidden under the static and global properties of the cells. These new approaches may provide us fresh cues to understand the cell response to ECM rigidity and protein composition.

REFERENCES

(1) Discher, D. E.; Janmey, P.; Wang, Y. L. Tissue cells feel and respond to the stiffness of their substrate. Science 2005, 310 (5751), 1139−1143. (2) Pelham, R. J., Jr.; Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (25), 13661−5. (3) Zajac, A. L.; Discher, D. E. Cell differentiation through tissue elasticity-coupled, myosin-driven remodeling. Curr. Opin. Cell Biol. 2008, 20 (6), 609−15. (4) Horejs, C. M.; Serio, A.; Purvis, A.; Gormley, A. J.; Bertazzo, S.; Poliniewicz, A.; Wang, A. J.; DiMaggio, P.; Hohenester, E.; Stevens, M. M. Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (16), 5908−13. (5) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 2006, 126 (4), 677−89.

K


Article

ACS Biomaterials Science & Engineering (6) Lee, J.; Abdeen, A. A.; Zhang, D.; Kilian, K. A. Directing stem cell fate on hydrogel substrates by controlling cell geometry, matrix mechanics and adhesion ligand composition. Biomaterials 2013, 34 (33), 8140−8. (7) Pek, Y. S.; Wan, A. C.; Ying, J. Y. The effect of matrix stiffness on mesenchymal stem cell differentiation in a 3D thixotropic gel. Biomaterials 2010, 31 (3), 385−91. (8) Rowlands, A. S.; George, P. A.; Cooper-White, J. J. Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation. American journal of physiology. Cell physiology 2008, 295 (4), C1037−44. (9) Indra, I.; Beningo, K. A. An in vitro correlation of metastatic capacity, substrate rigidity, and ECM composition. J. Cell. Biochem. 2011, 112 (11), 3151−8. (10) Kanchanawong, P.; Shtengel, G.; Pasapera, A. M.; Ramko, E. B.; Davidson, M. W.; Hess, H. F.; Waterman, C. M. Nanoscale architecture of integrin-based cell adhesions. Nature 2010, 468 (7323), 580−4. (11) Hoffman, B. D.; Grashoff, C.; Schwartz, M. A. Dynamic molecular processes mediate cellular mechanotransduction. Nature 2011, 475 (7356), 316−23. (12) Seong, J.; Tajik, A.; Sun, J.; Guan, J. L.; Humphries, M. J.; Craig, S. E.; Shekaran, A.; Garcia, A. J.; Lu, S.; Lin, M. Z.; Wang, N.; Wang, Y. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (48), 19372−7. (13) Rossier, O.; Octeau, V.; Sibarita, J. B.; Leduc, C.; Tessier, B.; Nair, D.; Gatterdam, V.; Destaing, O.; Albiges-Rizo, C.; Tampe, R.; Cognet, L.; Choquet, D.; Lounis, B.; Giannone, G. Integrins beta1 and beta3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat. Cell Biol. 2012, 14 (10), 1057−67. (14) Jiang, L.; Sun, Z.; Chen, X.; Li, J.; Xu, Y.; Zu, Y.; Hu, J.; Han, D.; Yang, C. Cells Sensing Mechanical Cues: Stiffness Influences the Lifetime of Cell-Extracellular Matrix Interactions by Affecting the Loading Rate. ACS Nano 2016, 10 (1), 207−17. (15) Plotnikov, S. V.; Pasapera, A. M.; Sabass, B.; Waterman, C. M. Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration. Cell 2012, 151 (7), 1513−27. (16) Plotnikov, S. V.; Sabass, B.; Schwarz, U. S.; Waterman, C. M. High-resolution traction force microscopy. Methods Cell Biol. 2014, 123, 367−94. (17) Liu, K.; Yuan, Y.; Huang, J.; Wei, Q.; Pang, M.; Xiong, C.; Fang, J. Improved-throughput traction microscopy based on fluorescence micropattern for manual microscopy. PLoS One 2013, 8 (8), e70122. (18) Tse, J. R.; Engler, A. J., Preparation of hydrogel substrates with tunable mechanical properties. In Current Protocols in Cell Biology; Wiley: New York, 2010; Chapter 10, Unit 10.16 ; DOI: 10.1002/ 0471143030.cb1016s47. (19) Wolfenson, H.; Iskratsch, T.; Sheetz, M. P. Early events in cell spreading as a model for quantitative analysis of biomechanical events. Biophys. J. 2014, 107 (11), 2508−14. (20) Skau, C. T.; Plotnikov, S. V.; Doyle, A. D.; Waterman, C. M. Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (19), E2447−56. (21) Butcher, D. T.; Alliston, T.; Weaver, V. M. A tense situation: forcing tumour progression. Nat. Rev. Cancer 2009, 9 (2), 108−22. (22) Li, J.; Chen, H.; Xu, Y.; Hu, J.; Xie, F. Q.; Yang, C. Integrin endocytosis on elastic substrates mediates mechanosensing. J. Biomech. 2016, 49 (13), 2644−2654. (23) Valdes, J. L.; Tang, J.; McDermott, M. I.; Kuo, J. C.; Zimmerman, S. P.; Wincovitch, S. M.; Waterman, C. M.; Milgram, S. L.; Playford, M. P. Sorting nexin 27 protein regulates trafficking of a p21-activated kinase (PAK) interacting exchange factor (beta-Pix)-G protein-coupled receptor kinase interacting protein (GIT) complex via a PDZ domain interaction. J. Biol. Chem. 2011, 286 (45), 39403−16. (24) Zeltz, C.; Gullberg, D. The integrin-collagen connection–a glue for tissue repair? J. Cell Sci. 2016, 129 (4), 653−64.

(25) Du, J.; Chen, X.; Liang, X.; Zhang, G.; Xu, J.; He, L.; Zhan, Q.; Feng, X. Q.; Chien, S.; Yang, C. Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (23), 9466− 71.

L


Quantitative Analyses of Dynamic Features of Fibroblasts on Different

Recommend Documents