A switchable light-responsive azopolymer conjugating protein micropatterns with topography for mechanobiological studies

Abstract

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However, conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Current methods to fabricate dynamic platforms usually rely on complex chemical strategies or require specialized apparatuses. Also, with these methods, the integration of dynamic signals acting on different length scales is not straightforward, whereas, in some applications, it might be beneficial to act on both a microscale level, that is, cell shape, and a nanoscale level, that is, cell adhesions. Here, we exploited a confocal laser-based technique on a light-responsive azopolymer displaying micropatterns of adhesive islands. The laser light promotes a directed mass migration and the formation of submicrometric topographic relieves. Also, by changing the surface chemistry, the surfacing topography affects cell spreading and shape. This method enabled us to monitor in a non-invasive manner the dynamic changes in focal adhesions, cytoskeleton structures, and nucleus conformation that followed the changes in the adhesive characteristic of the substrate. Focal adhesions reconfigured after the surfacing of the topography, and the actin filaments reoriented to coalign with the newly formed adhesive island. Changes in cell morphology also affected nucleus shape, chromatin conformation, and cell mechanics with different timescales. The reported strategy can be used to investigate mechanotransduction-related events dynamically by controlling cell adhesion at cell shape and focal adhesion levels. The integrated technique enables achieving a submicrometric resolution in a facile and cost-effective manner.

Keywords: azopolymer; cell adhesion; cell shape; dynamic substrates; light-responsive; mechanotransduction; nucleus shape.

FIGURE 1

(A) Schematic representation of the patterning process via laser scanning. (B) Fluorescence images of the micropatterned fluorescently labelled fibronectin island on a flat pDR1m substrate prior to pattern inscription and (C) on the same substrate after the inscription. Bar is 10 μm. (D) AFM image of a pDR1m substrate displaying the laser-written topographic pattern. (E) Representative height profile of a horizontal cross section of the topographic relieves.

FIGURE 2

(A) Immunofluorescence images of ASC52telo cells cultivated on the flat micropatterned pDR1m substrate (0 h) or on pDR1m displaying the topographic pattern 1, 4, or 24 h after the inscription. Nuclei (DAPI) are in blue, FAs (paxillin) are in green, and actin (phalloidin) is in red. Bar is 10 μm. Box-and-whiskers plot of the FA area (B) and FA orientation (C). The asterisks indicate that the median value of the distribution is significantly different from 45°.

FIGURE 3

(A) Confocal images of phalloidin-stained ASC52telo cells cultivated on the flat micropatterned pDR1m substrate (0 h) or on pDR1m displaying the topographic pattern 1, 4, or 24 h after the inscription. Bar is 10 μm. (B) Box-and-whiskers plot of the cell area, (C) aspect ratio, and (D) roundness. The asterisks indicate statistically significant differences between groups in (C), whereas they indicate that the median value of the distribution is significantly different from 45° in (D).

FIGURE 4

Box-and-whiskers plots of the nucleus aspect ratio (A) and the orientation of the nucleus major axis, with respect to the horizontal axis (B). Asterisks indicate significant differences between groups in (A), whereas they indicate that the median value of the distribution is significantly different from 45° in (B).

FIGURE 5

Fluorescence images of equatorial cross sections of nuclei of cells cultivated on (A) flat pDR1m substrates or (B) 24 h after the inscription of the topographic pattern. DAPI-rich spots are in blue, and H3K9Ac-rich spots are in green. Bar is 5 μm. (C) Box-and-whiskers plot of the volumes of the DAPI (blue) or H3K9Ac (green) spots. (D) Bar chart of the overall fluorescence intensity of the DAPI or (D) H3K9Ac signal. Asterisk indicates a significant difference with respect to the 0 h case.

FIGURE 6

Time variation of Young’s modulus of the nuclear region normalized with respect to the modulus evaluated prior to pattern inscription (at time t = 0-).