Chromatin de-condensation by switching substrate elasticity

Abstract

Mechanical properties of the cellular environment are known to influence cell fate. Chromatin de-condensation appears as an early event in cell reprogramming. Whereas the ratio of euchromatin versus heterochromatin can be increased chemically, we report herein for the first time that the ratio can also be increased by purely changing the mechanical properties of the microenvironment by successive 24 h-contact of the cells on a soft substrate alternated with relocation and growth for 7 days on a hard substrate. An initial contact with soft substrate caused massive SW480 cancer cell death by necrosis, whereas approximately 7% of the cells did survived exhibiting a high level of condensed chromatin (21% heterochromatin). However, four consecutive hard/soft cycles elicited a strong chromatin de-condensation (6% heterochromatin) correlating with an increase of cellular survival (approximately 90%). Furthermore, cell survival appeared to be reversible, indicative of an adaptive process rather than an irreversible gene mutation(s). This adaptation process is associated with modifications in gene expression patterns. A completely new approach for chromatin de-condensation, based only on mechanical properties of the microenvironment, without any drug mediation is presented.

Figure 1

Conditions and timing of the experiments.

Figure 2

Cell survival and vinculin assembly are influenced by switching the substrate elasticity. (A) Percentage of surviving SW480 cells based on the S1 from three pooled independent experiments (error bars represent s.e.m.). (B) Representative images of cells cultured for 24 h on glass, 1^st E₂₀, 2^nd E₂₀ and 3^rd E₂₀ immunolabelled with anti-vinculin counterstained with Hoechst 33258 and phalloidin. Scale bars: 10 µm. (C) Quantitative data for cell areas (in µm²), based on B, were determined using ImageJ. We used 200–300 cells for each condition (glass, 1^st E₂₀, 2^nd E₂₀ and 3^rd E₂₀). (D) Quantitative data for nuclear areas (in µm²) based on B were determined using ImageJ. We used 200–300 cells for each condition. (E) Quantification of vinculin spots (in µm) based on B by measuring 20 contacts per cell in 20 cells for each condition. (F) Quantification of actin stress fibers (in µm) using ImageJ based on B by measuring the length of 20 fibers per cell in 20 cells for each condition. (C–F) Results for three independent experiments are shown (error bars represent s.e.m.).

Figure 3

Cell motility influenced by switching the substrate elasticity. Motility of SW480 cells after successive seeding on soft substrate. (A) Time-lapse monitoring representative images of cells cultured on glass, 2^nd E₂₀ and 3^rd E₂₀ for 20 h. Inset: enlargement of the white dotted panel. Arrowhead: coloured cellular trajectory. (B) Cell trajectories from A by NIS-Elements 3D tracking. (C) Number of cells analysed, percentage of cells in class 1 and class 2, and mean speed of these cells, from A. The results from two pooled independent experiments for each condition.

Figure 4

Chromatin de-condensation by switching the substrate elasticity (A) Representative ultrastructural images of cells after 24 h of culture on 1^st glass, 1^st E₂₀, 2^nd E₂₀, 3^rd E₂₀, 4^th E₂₀, and 4^th glass. Scale bars: 5 µm, arrow: heterochromatin, star: euchromatin, (B) Percentage of heterochromatin in the nucleus of approximately 20 cells for each condition, from A, for two pooled independent experiments (error bars represents s.e.m.).

Figure 5

Cell survival is chromatin conformation-dependent (A) Representative ultrastructural images of cells after 24 h of culture on the 1^st E₂₀ + TSA, 2^nd E₂₀ + TSA and 3^rd E₂₀ + ANA. Scale bars: 5 µm, arrow: heterochromatin, star: euchromatin. (B) Percentage of heterochromatin in the nucleus of approximately 20 cells for each condition, from A, for two pooled independent experiments (error bars represents s.e.m.). (C) Percentage of surviving SW480 cells from S4 for two or three pooled independent experiments (error bars represent s.e.m.). (D) Representative images of cells cultured for 24 h on 1^st E₂₀ + TSA, 2^nd E₂₀ + TSA and 3^rd E₂₀ + ANA immunolabelled with anti-vinculin andcounterstained with Hoechst 33258 and phalloidin. Scale bars: 10 µm. (E) Quantification of vinculin spots (in µm) based on D by measuring 20 contacts per cell in 20 cells for each condition. (F) Quantification of actin stress (in µm) using ImageJ based on D by measuring the length of 20 actin stress fibers per cell in 20 cells for each condition. (D–F) Results for three independent experiments (error bars represent s.e.m.).

Figure 6

Cell motility is chromatin conformation-dependent. (A) Time-lapse monitoring representative images of cells cultured on 1^st E₂₀ + TSA, 2^nd E₂₀ + TSA and 3^rd E₂₀ + ANA for 20 h. Inset: enlargement of the white dotted panel, arrowhead: coloured cellular trajectory. (B) Cell trajectories, from A, by NIS-Elements 3D tracking. (C) Number of cells analysed, percentage of cells in class 1 and class 2 and mean speed of these cells, from A. The Results from two pooled independent experiments for each condition. Class 1 speed of 1^st E₂₀ + TSA vs 2^nd E₂₀ + TSA vs 3^rd E₂₀ + ANA: **.

Figure 7

Reversible adaptation of chromatin de-condensation. (A) Representative images acquired by phase contrast microscopy: 1^st E₂₀ cells cultured for 24 h on E₂₀. Yellow arrowhead: refractive objects corresponding to surviving cells; E₂₀ cells cultured on the 1^st E₂₀ for 24 h and transferred to new E₂₀ for 24 h without an intermediate step on glass. Red arrowhead: non-refractive objects corresponding to necrotic cells. Scale bars: 100 µm. (B) Percentage of surviving SW480 cells from S4D for two independent experiments (the error bars represent the s.e.m.). (C) Representative ultrastructural images of cells cultured on 1^st E₂₀ - glass, 2^nd E₂₀ - glass, 3^rd E₂₀ - glass and 4^th E₂₀ - glass. Scale bars: 5 µm. (D) Percentage of heterochromatin in the nucleus of approximately 20 cells for each condition from C for two pooled independent experiments (error bars represents s.e.m.).

Figure 8

Functional clustering annotation of the differentially expressed genes. (A) Seven clusters identified with DAVID 6.8 with an enrichment score >2. (B) Cartoon summarizing the influence of the number of hard/soft substrate SW480 cell relocation on survival and chromatin conformation (hard = glass, soft = E₂₀).