Contribution of actin filaments and microtubules to cell elongation and alignment depends on the grating depth of microgratings
© Lee et al. 2016
Received: 27 November 2015
Accepted: 19 April 2016
Published: 29 April 2016
It has been reported that both chemical and physical surface patterns influence cellular behaviors, such as cell alignment and elongation. However, it still remains unclear how actin filament and microtubules (MTs) differentially respond to these patterns.
We examined the effects of chemical and physical patterns on cell elongation and alignment by observing actin filament and MTs of retinal pigment epithelium-1(RPE-1) cells, which were cultured on either fibronectin (FN)-line pattern (line width and spacing: 1 μm) or FN-coated 1 μm gratings with two different depths (0.35 or 1 μm). On the surface with either FN-line pattern or micrograting structure, the cell aspect ratios were at least two times higher than those on the surface with no pattern. Cell elongation on the gratings depended on the depth of the gratings. Cell elongation and alignment on both FN-line pattern and 1 μm gratings with 0.35 μm depth were perturbed either by inhibition of actin polymerization or MT depletion, while cell elongation and alignment on 1 μm gratings with 1 μm depth were perturbed only by MT depletion.
Our results suggest that the contribution of actin filaments and MTs to the elongation and alignment of epithelial cells on microgratings depends on the groove depth of these gratings.
Cells respond to chemical and physiological cues of various extracellular environments in vitro or in vivo. Cells integrate these various cues and generate appropriate cellular responses, such as alignment, migration, and elongation [1, 2]. Chemical cues are mediated by a specific integrin-ligand binding . On the other hand, physical cues are generated by various topographic structures, such as gratings (anisotropic grooves and ridges), cliffs, pillars, and islands . Only anisotropic topographic structures can induce different types of cells to align and elongate along the direction of axis. This phenomenon is called contact guidance which has been marked especially when the grooves or ridges are narrower than cells on the microgrooves [5–7]. It was hypothesized that contact guidance is a consequence of a cell’s ability to spread in the direction of higher rigidity, which is the length of the anisotropic structures [8, 9].
In previous studies, there have been significant differences in the cytoskeletal organization of cells located between topographic and flat surfaces [6, 10, 11]. When cells are cultured on topographic surfaces, actin filaments [10, 11] and microtubules (MTs)  align along the grooves and ridges, but these cytoskeletons do not show any preferential orientations on a flat surface. With alignment, actin filaments and MTs also play an important role in regulating cell length [12, 13] and cell spreading [14–16]. The cortical tension is mediated by actin and myosin. This tension plays a pivotal role in regulating cell shape and spreading . The dynamics of MTs also influences the regulation of cell length and cell shape in fission yeast . However, it has not yet to be clearly understood how cytoskeletons get differentially organized in the presence of different surfaces of topographical structures. For example, cell alignment and elongation are dependent on aspect ratio (depth/width) of anisotropic structures [8, 9], but it still remains unclear how actin filaments and microtubules (MTs) differentially respond to anisotropic structure with different aspect ratios.
In this study, we used a live-cell imaging technique to meticulously observe the alignment and elongation of non-transformed human retinal pigment epithelial (RPE-1), which are cultured on chemically or physically patterned anisotropic surfaces. Most of cancer cell lines are aneuploid conditions, which are caused by various mechanisms, including impaired microtubule dynamics . Since we wanted to see the role of MTs on contact guidance, we chose RPE-1, which is one of the few cell lines that can maintain a diploid karyotype over an extended culture period. Using RPE-1, it has been determined how chemical patterns affect the shape and division of cells [17, 20]. In this study, RPE-1 cells were cultured in different substrates: tissue culture polystyrene (TCPS), TCPS printed with fibronectin (FN) lines (line width and spacing: 1 μm), and 1 μm polydimethylsiloxane (PDMS) gratings (0.35 or 1 μm depth) coated with FN. The aspect ratios (length/width) and alignment of cells on these substrates were subsequently investigated to determine how FN geometry and anisotropic surface topography affected contact guidance behaviors such as cell alignment and elongation. In addition, the actin filament and microtubule inhibitors, cytochalasin D (CD) and nocodazole (Noc), respectively, were used to investigate the roles of actin filaments and MTs on the behaviors.
Results and discussion
RPE-1 cells align and elongate along the direction of FN line or microgratings
Effect of different substrates on cell alignment
FN-linesa (1 µm line)
1 µm gratingb (0.35 µm deep)
1 µm gratingc (1 µm deep)
Cell alignment (°)d
10.7 ± 7.7 (n = 106)e
8.4 ± 7.4 (n = 103)
3.8 ± 3.0 (n = 133)
For cell length analysis, the cell aspect ratio (R) was regarded as parameter at a fixed time point. R was defined as a ratio of cell length (L) to cell width (W). R values indicate that cells on FN-lines and 1 µm gratings (0.35 and 1 µm deep) were significantly more elongated than those on the flat surface of TCPS (Fig. 1b; Table 3). Cells on 1 µm gratings (1 µm depth) displayed the highest R values (Fig. 1b; Table 3) among the cells on the patterns (FN-lines and 1 µm gratings (0.35 and 1 μm deep). Wong et al.  reported that cell elongations on microngratings (1, 2 and 10 μm) decreased on wider gratings. Crouch et al.  reported a similar trend. It was also reported that compared to cells on microgratings, those on 100 nm gratings were less aligned to the direction of the grating axis . Due to the reason, we chose 1 and 2 μm gratings. Cells on 2 µm gratings (2 µm depth) were slightly longer than those on 1 µn grating (1 µm depth) (Additional file 1: Figure S2), which is in accordance with the previous report . However, cytoskeleton images of cells on 2 µm gratings were of poor quality because the gratings caused diffraction of light. Thus, we used 2 µm gratings only for cell alignment and elongation studies that do not require high resolution images. Since FN-lines were prepared by selectively coating TCPS surface with FN, they were considered as gratings with zero aspect ratio. The results (Fig. 1b; Table 3) indicate that cell elongation was dependent on grating aspect ratio (depth/width). Similar reports were found elsewhere [8, 9]. The cytoskeleton of cells on 1 µm gratings (1 µm depth) would have to be bended at a higher degree in order to conform to the grooves, compared to that of cells on either 1 µm gratings (0.35 µm depth) or FN-lines. Based on this explanation, cells on the deep gratings are less likely to spread in the lateral direction than those on both shallow gratings (0.35 µm depth) and FN-lines. Further studies on filopodium and lamellapodium formation on gratings are needed to support the explanation.
Cell alignment and elongation on 1 µm gratings with 1 µm depth are not interfered by depletion of actin filaments
Effect of drug treatments on cell alignment of different substrates
Cell alignment (°)a
FN-lines (1 µm line)
1 µm grating (0.35 µm deep)
1 µm grating (1 µm deep)
10.7 ± 7.7 (n = 106)
8.4 ± 7.4 (n = 103)
3.8 ± 3.0 (n = 133)
10.0 ± 8.6 (n = 112)
8.7 ± 8.4 (n = 100)
3.9 ± 3.0 (n = 109)
20.2 ± 16.5 (n = 108)
8.5 ± 7.5 (n = 104)
3.8 ± 3.0 (n = 104)
28.2 ± 17.2 (n = 106)
17.4 ± 14 (n = 104)
2.6 ± 1.9 (n = 101)
21.7 ± 15.6 (n = 103)
18.4 ± 17.9 (n = 110)
6.8 ± 4.8 (n = 103)
25.6 ± 16.3 (n = 110)
23.5 ± 19.4 (n = 108)
24.0 ± 18.3 (n = 104)
Effect of drug treatments on cell aspect ratio (R) of different substrates
Cell aspect ratio (R)a
FN-lines (1 µm line)
1 µm grating (0.35 µm deep)
1 µm grating (1 µm deep)
2.5 ± 0.7 (n = 153)
5.0 ± 1.3 (n = 141)
4.8 ± 1.3 (n = 111)
6.3 ± 1.5 (n = 123)
2.2 ± 0.8 (n = 124)
3.8 ± 1.3 (n = 148)
3.5 ± 1.3 (n = 101)
6.1 ± 1.9 (n = 156)
2.1 ± 0.6 (n = 149)
3.3 ± 1.1 (n = 154)
3.0 ± 1.2 (n = 104)
5.9 ± 1.8 (n = 162)
2.0 ± 0.7 (n = 155)
2.8 ± 0.9 (n = 131)
3.0 ± 1.1 (n = 105)
9.1 ± 2.8 (n = 182)
1.7 ± 0.5 (n = 149)
1.8 ± 0.6 (n = 102)
3.5 ± 1.0 (n = 123)
5.9 ± 1.3 (n = 112)
1.5 ± 0.4 (n = 112)
1.5 ± 0.4 (n = 102)
1.8 ± 2.1 (n = 102)
1.7 ± 0.4 (n = 110)
Our results (Fig. 3) indicate that cells on 1 µm gratings (1 µm depth) were elongated when they were treated with CD. Compared to MTs that were not treated with CD on any gratings, the MTs were more condensed and aligned along to the ridges of 1 µm deep gratings when they were treated with 1 µm of CD (Fig. 4; Additional file 1: Figure S4). This hinted that unlike MTs on the shallow gratings those on the deep gratings were not able to be randomly orientated. These results suggest that the deep groove substratum compensates for the actin filament deficiency by fully enforcing condensation and elongation on aligned microtubules.
Cell alignments on 1 µm deep gratings are interfered only by depletion of MT
In this study, we investigated how chemical and physical anisotropies on the surface affected cell alignment and elongation in RPE-1 cells using μCP and microfabrication. Moreover, we also verified that cell elongation strongly depends on the depth of microgratings. Previously, the relative importance of actin filaments and MTs in response to the anisotropic structures has not been determined. Our results indicate that neither cortical actin filament nor FA is a prerequisite for alignment and elongation of cells on 1 µm gratings (1 µm depth) and 2 ratings (2 μm depth). Furthermore, MTs dynamics were imposed by geometrical constraints, such as depth. These parameters played an essential role in the alignment and elongation of non-transformed RPE-1 cells.
The influence of microgratings on cell elongation and alignment is routinely observed in many different cell types including normal fibroblast cells. However, these microgratings may have a different effect on cancer epithelial cells that have impaired MT stabilities. Thus, these studies with non-transformed epithelial cells would determine whether deregulated behaviors can contribute to carcinogenesis. In addition to our basic understanding of cellular response and anisotropic surface structures, our findings provide hints for the application of synthetic structures, which are used for the differentiation of stem cells. Micro- and nanogratings are known to enhance neural differentiation of stem cells through cell elongation [23, 24].
Preparation of FN-line pattern and microgratings
1 μm gratings (1 μm ridge width, 2 μm pitch) having two different groove depths (0.35 or 1 μm); these gratings were developed using PDMS (polydimethylsiloxane) (Sylgard® 184, Dow Corning) by soft lithography . In a 4-inch silicon wafer, we fabricated master molds with grating structures using standard photolithography. After successfully conducting the fabrication process, we performed a reactive ion etching process. For this purpose, 10 mL of PDMS and its curing agent were mixed in a 10:1 ratio (w/w). The resultant mixture was then poured into each mold and cured at 65 ℃ for 2 h. To improve cell adhesion on the grating structure, the gratings were coated with 2 µg/mL solution of FN (Sigma Chemical. Co.) at 37 °C for 1 h.
The FN-line pattern was prepared on TCPS by microcontact printing (μCP) . In this process, a PDMS stamp with gratings (1 μm ridge width, 2 μm pitch, 1 μm depth) was immersed for 1 h into a solution containing Cy3.5-labeled FN (final conc. 0.2 mg/mL) (GE Healthcare) and bare FN (final conc.16 µg/mL). Then, the stamp was air-dried for 30 min at room temperature before printing FN onto TCPS. In the meantime, the TCPS surface was oxidized in a plasma chamber (Harrick Plasma, Ithaca, NY, USA) for 3 min. The PDMS stamp was carefully placed onto a freshly cleaned TCPS for 30 min. As soon as the stamp was removed from the TCPS surface, we poured 0.2 % Pluronic F-127 (Sigma Chemical Co.) in phosphate-buffered saline (PBS) into TCPS. Then, we incubated it for 1 h at room temperature to make the uncoated area passive. Before cell seeding, TCPS was washed twice with PBS.
RPE-1 (ATCC #CRL-4000) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Pittsburgh, PA, USA), which was supplemented with 10 % fetal bovine serum (FBS). Stock cell cultures were maintained in a humidified incubator at 37 °C in an atmosphere with 5 % CO2.
We added cytochalasin D (CD) (0.1–1 μm), actin polymerization inhibitor, or nocodazole (Noc) (0.04–1 μm), a MT polymerization inhibitor, right after cell seeding. CD and Noc were purchased from Sigma Chemical Co.
For indirect immunofluorescence microscopy, cover glass cultures were washed with PBS, fixed with 4 % paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and permeabilized with 0.5 % Triton X-100 in PBS as previously detailed . In this process, mouse anti-α-tubulin (Cell Signaling Technology, Seoul, Korea) and mouse anti-vinculin (Sigma Chemical Co.) were used as primary antibody (1:1000 dilution), while fluorescein isothiocyanate (FITC)-conjugated anti-mouse (Sigma Chemical Co.) was used as the secondary antibody (1:5000 dilution). For F-actin staining, cells were incubated with 5 µg/mL of tetramethyl rhodamine B isothiocyanate (TRITC)-phalloidin (Sigma Chemical Co.) in PBS for 30 min. Image stacks were acquired and de-convoluted using a Delta Vision System (GE Healthcare) centered on an IX70 inverted microscope (Olympus), which was equipped with a CoolSNAP HQ2 charge-coupled device camera (Photometrics, Tucson, AZ, USA).
Live cell microscopy and quantification
For live-cell imaging, cells were placed on either flat surface of TCPS, FN line pattern, or 1 µm gratings (0.35 or 1 µm deep) and incubated for 1 h. Time-lapse images were captured every 5 min for 24 h at 37 °C using Biostation CT (Nikon). Time-lapse image sequences were compiled and measured using Image J (NIH, USA). The cell aspect ratio (R) was determined by measuring cell length (L) and cell width (W) at 12 h after seeding. In this case, L is defined as the length of the long axis of the cell, while W is defined as the maximum width of a cell axis that is perpendicular to the long axis . Cell alignment is defined as the angular difference between the major axis of cells and the long axis of FN-line or each micrograting and was measured at 12 h after seeding with and without CD or Noc treatment. Cells with alignment angle less than 15° are deemed to be aligned .
Every experiment was conducted twice or three times on different days. All the data were expressed as mean ± SD. Data were analyzed using one-way ANOVA and a Bonferroni post hoc test. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA) and a two-tailed hypothesis. P values <0.01 were considered to be statistically significant.
SP organized and planned the project; KL involved all experiments; KL, EHK, NO, SP, NAT, and EKY fabricated microgratings. NHB, SJL, and KGL performed AFM. CYE performed 3D imaging. KL and SP analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
This work was equally supported by the Technology Innovation Program (#10050154, Business Model Development for Personalized Medicine Based on Integrated Genome and Clinical Information) funded by the Ministry of Trade, Industry & Energy and the Public Welfare and Safety Research program (#2012M3A2A1051681) through National Research Foundation (NRF) in Korea. EHK’s internship at Mechanobiology Institute (MBI) was financially supported by BK (Brain Korea) 21 program through NRF in Korea and MBI at National University of Singapore.
The authors declare that they have no competing interests.
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