Expression of vimentin alters cell mechanics, cell-cell adhesion, and gene expression profiles suggesting the induction of a hybrid EMT in human mammary epithelial cells

Abstract

Vimentin is a Type III intermediate filament (VIF) cytoskeletal protein that regulates the mechanical and migratory behavior of cells. Its expression is considered to be a marker for the epithelial to mesenchymal transition (EMT) that takes place in tumor metastasis. However, the molecular mechanisms regulated by the expression of vimentin in the EMT remain largely unexplored. We created MCF7 epithelial cell lines expressing vimentin from a cumate-inducible promoter to address this question. When vimentin expression was induced in these cells, extensive cytoplasmic VIF networks were assembled accompanied by changes in the organization of the endogenous keratin intermediate filament networks and disruption of desmosomes. Significant reductions in intercellular forces by the cells expressing VIFs were measured by quantitative monolayer traction force and stress microscopy. In contrast, laser trapping micro-rheology revealed that the cytoplasm of MCF7 cells expressing VIFs was stiffer than the uninduced cells. Vimentin expression activated transcription of genes involved in pathways responsible for cell migration and locomotion. Importantly, the EMT related transcription factor TWIST1 was upregulated only in wild type vimentin expressing cells and not in cells expressing a mutant non-polymerized form of vimentin, which only formed unit length filaments (ULF). Taken together, our results suggest that vimentin expression induces a hybrid EMT correlated with the upregulation of genes involved in cell migration.

Keywords: Twist1; cell-cell adhesion; desmoplakin; hybrid/partial EMT; intercellular forces; intracellular mechanics; vimentin.

FIGURE 1

Expression and assembly stages of vimentin in inducible MCF7. (A) Immunofluorescence images of vimentin expression in (A) (i) uninduced MCF7 and (ii) cumate-induced MCF7 cells expressing WT vimentin and (iii) ULF mutant vimentin. Magnified images of WT vimentin or ULF mutant expressing cells are shown in (iv) and (v) respectively. (B) Western blot showing the expression levels of vimentin in uninduced MCF7 cells, WT vimentin in MCF7 cells following induction and ULF mutant vimentin in MCF7 cells, run along with an internal control, GAPDH. (C) Immunofluorescence images of vimentin in cumate-induced MCF7 cells expressing WT vimentin. C (i) and C (ii) show the vimentin on day 1 (C-i) and day 2 (C-ii) post cumate induction as particles and squiggles respectively, indicating the assembly stages of vimentin. c-iii is observed on day 5 post cumate induction where vimentin is observed as an extensive filamentous network. Scale bar is 10 μm.

FIGURE 2

Vimentin expression does not alter the endogenous expression of keratin in MCF7. Immunofluorescence images of keratin 18 (A, D and G) and vimentin (B, E and H) in uninduced MCF7 cells (A–C), WT vimentin in induced MCF7 cells (D–F) and ULF mutant vimentin in MCF7 cells (G–I). Figures C, F and I show the overlay of keratin 18 and vimentin in the respective cells. Scale bar is 10 μm. (J) Graph showing the number of keratin junctions in uninduced MCF7 and in cells expressing either WT vimentin or ULF mutant vimentin. (K) Graph showing the keratin bundle thickness in uninduced MCF7 cells and following the induction of WT vimentin or ULF mutant vimentin. (L) Western blot showing the expression levels of keratin 8/18 in uninduced MCF7, MCF7 cells expressing WT vimentin or ULF mutant vimentin. GAPDH is used as loading control. One-way ANOVA was used to compare the differences between the groups. ****p < 0.0001. All the experiments were done on cells sorted for GFP expression 5 days post induction with cumate.

FIGURE 3

Expression of vimentin in MCF7 disrupts desmosomes. Immunofluorescence images of desmoplakin (A,D,G) and vimentin (B,E,H) in uninduced MCF7 (A,B,C), cumate-induced MCF7 expressing WT vimentin (D,E,F) or ULF mutant vimentin (G,H,I). Figures C, F and I show the overlay of desmoplakin and vimentin in the respective cells. Scale bar is 10 μm. Structured Illumination Microscopy (SIM) images of desmoplakin (J, N, R), vimentin (K, O, S), and keratin (L, P, T) in uninduced MCF7 cells (J–M), cumate-induced MCF7 cells expressing WT vimentin (N–Q) and cumate-induced MCF7 cells expressing ULF mutant vimentin (R–U). Figure M, Q and U show the respective overlays. Scale bar is 1 μm. (V) Graph showing the percentage of cells having disrupted desmoplakin in uninduced MCF7 cells (N = 205) and in MCF7 cells induced to express either WT (N = 101) or ULF mutant vimentin (N = 191). One way ANOVA was used to compare the differences between the groups.****p < 0.0001; ***p < 0.001. All the experiments were done on cells sorted for GFP expression 5 days post induction with cumate (Des–desmoplakin; Vim–vimentin; Ker–keratin).

FIGURE 4

Vimentin expression reduced traction and intercellular forces in MCF7. (A) Representative phase contrast images of individual islands of uninduced MCF7 cells, and cumate-induced MCF7 cells expressing either WT vimentin or ULF mutant vimentin (scale bar 200 μm). (B) Color maps of magnitude of cell-substrate traction of cell islands corresponding to the images in column (A). (C) Color maps of intercellular tension of the cell islands (D) Root-mean-square (rms) traction among uninduced MCF7 cells (n = 14) and MCF7 cells expressing either WT vimentin (n = 21) or ULF mutant vimentin (n = 15). (E) Average of intercellular stress comparison among uninduced MCF7 cells (n = 14), cumate-induced MCF7 cells expressing WT vimentin (n = 21) or ULF mutant vimentin (n = 15). In panels D and E, each dot represents an independent cell island, horizontal lines indicate means, and ** indicates p < 0.01 compared to uninduced (ANOVA with Bonferroni correction). (F) Intercellular stress plotted against normalized radial position, where 0 indicates the center of the cell island and R ₀ is the radius of the cell island. Each line represents a different cell island.

FIGURE 5

Vimentin expression reduces intracellular motion. (A) Trajectories of particles measured in 10 different cells in each group, from videos taken every 12 ms over 20 s time intervals. Scale bar, 0.5 µm. (B) Mean Square Displacement (MSD) of particles. (C) Compliance of cytoplasm. ${\delta }^2$ is the MSD over 12 ms, $k_b$ is Boltzmann constant, $T=$ 310 K is temperature, $d=0.5$ µm is the diameter of the particles. One way ANOVA was used to compare the differences between the groups. ****p < 0.0001.

FIGURE 6

Upregulation of TWIST1 requires WT vimentin. Circos plot representation of significantly enriched pathways linked with (A) WT vimentin vs. uninduced; (B) ULF mutant vimentin vs. uninduced; (C) WT vimentin vs. ULF mutant vimentin. The ribbon/arc that originates from different genes and terminates at associated pathways demonstrates the connectivity of genes and overrepresented pathways; (D) Heatmap shows the fold change values of transcription factors and few of the mesenchymal markers in cells expressing WT or ULF mutant vimentin. (E) Graph depicts the relative fold change values of genes obtained by real time PCR (n = 3). 18S rRNA was used as the internal control.