Keratins significantly contribute to cell stiffness and impact invasive behavior

Abstract

Cell motility and cell shape adaptations are crucial during wound healing, inflammation, and malignant progression. These processes require the remodeling of the keratin cytoskeleton to facilitate cell-cell and cell-matrix adhesion. However, the role of keratins for biomechanical properties and invasion of epithelial cells is only partially understood. In this study, we address this issue in murine keratinocytes lacking all keratins on genome engineering. In contrast to predictions, keratin-free cells show about 60% higher cell deformability even for small deformations. This response is compared with the less pronounced softening effects for actin depolymerization induced via latrunculin A. To relate these findings with functional consequences, we use invasion and 3D growth assays. These experiments reveal higher invasiveness of keratin-free cells. Reexpression of a small amount of the keratin pair K5/K14 in keratin-free cells reverses the above phenotype for the invasion but does not with respect to cell deformability. Our data show a unique role of keratins as major players of cell stiffness, influencing invasion with implications for epidermal homeostasis and pathogenesis. This study supports the view that down-regulation of keratins observed during epithelial-mesenchymal transition directly contributes to the migratory and invasive behavior of tumor cells.

Keywords: Boyden chamber; cell mechanics; intermediate filaments; optical stretcher.

Fig. 1.

Keratins are major determinants of cell stiffness. (A) (Left) Schematic of the µOS setup showing a strongly deforming KO cell treated with LatA. (Right) Laser pattern used for all µOS measurements. (B and C) Data sets from optical stretcher measurements of keratin-free and K5/K14 reexpressing cells compared with WT cells. Characterized by creep deformation curves J(t) (B) and J (t = 3 s) (C) (***P < 0.001). (D) Immunofluorescence analysis using keratin and tubulin antibodies and phalloidin staining (actin). (E) Keratin 5, tubulin, and actin expression detected by Western blotting of total protein lysates. (Scale bar, 10 μm.)

Fig. 2.

LatA treatment enhances cell softness. (A) µOS data of WT, KO, and K5 cells in comparison with LatA-treated cells and respective cell counts (***P < 0.001; **P < 0.01; *P < 0.05). (B) Normalized creep curves of LatA treated and untreated cells (Inset). (C) LatA-treated cells immunostained for cytoskeletal proteins keratin 5, α-tubulin, and actin (phallodin). (Scale bar, 10 μm.)

Fig. 3.

Loss of keratins promotes invasion. (A) Quantification of transwell invasion experiments. The mean number of invaded cells per transwell membrane is presented. (B) Proliferation assay of keratinocytes following Ki67 staining. Error bars represent SD of three independent experiments. (C) Immunofluorescence analysis of cells having completed invasion at the bottom of the transwell membrane stained for vinculin at day 4 and day 7 (***P < 0.001 **P < 0.01). (Scale bar, 10 μm.)

Fig. 4.

Increased formation of colonies in keratin-free cells. (A) Formation of colonies surrounded by Matrigel. (B) Phase contrast images of cells growing in a 3D environment. (C) Colony assay stained using DAPI (***P < 0.001). (Scale bar, 100 μm.)