Keratins significantly contribute to cell stiffness and impact invasive behavior Kristin Seltmanna,1, Anatol W. Fritschb,1, Josef A. Käsb,2, and Thomas M. Magina,2 aTranslational Center for Regenerative Medicine and Institute of Biology, and bFakultät für Physik und Geowissenschaften, Abteilung Physik der Weichen Materie, Universität Leipzig, 04103 Leipzig, Germany Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved August 9, 2013 (received for review June 5, 2013) Cell motility and cell shape adaptations are crucial during wound which include active cell shape changes and increasing cell softness healing, inflammation, and malignant progression. These processes in tumor cells, rely largely on cell deformability as cells have require the remodeling of the keratin cytoskeleton to facilitate cell– to transmigrate their surrounding tissue (5–7). cell and cell–matrix adhesion. However, the role of keratins for bio- For small deformations imposed on cells, cortical actin is mechanical properties and invasion of epithelial cells is only partially generally seen as the dominant contributor to cell stiffness (8– understood. In this study, we address this issue in murine keratino- 10). Surprisingly, MTs are reported to have little or no significant cytes lacking all keratins on genome engineering. In contrast to effect on cell stiffness in this regime (11, 12). Based on the visco- predictions, keratin-free cells show about 60% higher cell deform- elastic properties of IFs in vitro, it was suggested that they might ability even for small deformations. This response is compared with be important for cell integrity at large deformations (13). Sup- the less pronounced softening effects for actin depolymerization fi induced via latrunculin A. To relate these findings with functional porting this hypothesis, studies on vimentin-de cient cells revealed consequences, we use invasion and 3D growth assays. These experi- that they were less stiff than WT cells at high stresses, whereas for ments reveal higher invasiveness of keratin-free cells. Reexpres- low stresses, there seemed to be no significant influence (14). Until sion of a small amount of the keratin pair K5/K14 in keratin-free now, the contribution of keratins to cell stiffness has not been cells reverses the above phenotype for the invasion but does not fully examined. with respect to cell deformability. Our data show a unique role of To investigate the overall contribution of the keratin IF cy- keratins as major players of cell stiffness, influencing invasion toskeleton to cell stiffness and invasive behavior, we generated BIOPHYSICS AND with implications for epidermal homeostasis and pathogenesis. This keratinocyte cell lines devoid of the entire keratin cytoskeleton, COMPUTATIONAL BIOLOGY study supports the view that down-regulation of keratins observed together with a rescue cell line reexpressing the single keratin – during epithelial mesenchymal transition directly contributes to the pair K5/K14 (3, 15). Based on measurements in a microfluidic migratory and invasive behavior of tumor cells. optical stretcher analyzing the noncontact deformability of sta- tistically relevant numbers of keratin-deficient, WT, and rescue cell mechanics | intermediate filaments | optical stretcher | Boyden chamber cells (16), we identify a major contribution of the keratin cyto- PHYSICS skeleton to whole cell deformability, which we show is significantly greater than that of actin for small deformations. Moreover, we ell shape and tissue integrity largely depend on the cyto- Cskeleton, which in mammals is composed of actin filaments, show using a Boyden chamber assay and 3D tissue culture that loss microtubules (MTs), and intermediate filaments (IFs). Together, of keratins enhances invasive behavior of keratinocytes, which can they provide resilience against force, coordinate cell motility, fi mediate intracellular movements, and are involved in cell sig- Signi cance naling (1, 2). Although mechanical resilience is of importance for the integrity of whole tissues, it is reasonable that for processes In many processes such as wound healing, inflammation, and such as migration of cells out of dense tissues, single cells need to cancer progression, the cytoskeleton is influencing cell motility be highly deformable. The role of actin filaments and MTs is and cell shape. Thus far, in contrast to the actin and microtu- relatively well understood. However, the understanding of the bule cytoskeleton, intermediate filament proteins are not well respective contribution of IFs, encoded by a gene family of >70 investigated in this context. Here, we show that keratin-free members in humans and mice, to cell deformability, migration, cells from mice skin lacking all keratins on genome engi- and signaling is only beginning to emerge (2). neering have about 60% higher cell deformability even for The IF cytoskeleton of epithelia is composed of keratins that small deformations in contrast to a smaller effect generated assemble from heterodimers of at least one type I and type II by actin depolymerization. Keratin-free cells are more invasive keratin into long, apolar IFs. It is believed that the cell type– and and show an increased growth in a 3D assay. Our study differentiation state–specific expression of keratin isotypes con- highlights keratins’ role in cell stiffness and its influence in tributes significantly to micromechanical properties of epithelial invasion, supporting the view that down-regulation of kera- – tissues (2). Basal epidermal keratinocytes express the keratin tins observed during epithelial mesenchymal transition di- pair K5/K14, which are replaced by K1/K10 during skin differ- rectly contributes to the migratory and invasive behavior of entiation, whereas tissue regeneration is accompanied by tran- tumor cells. sient K6/K16 up-regulation and repression of K1/K10 (3). The Author contributions: K.S., A.W.F., J.A.K., and T.M.M. designed research; K.S. and A.W.F. occurrence of cell fragility diseases caused by keratin mutations, performed research; K.S. and A.W.F. contributed new reagents/analytic tools; K.S. and followed by collapse of the keratin cytoskeleton, has suggested A.W.F. analyzed data; and K.S., A.W.F., J.A.K., and T.M.M. wrote the paper. a major contribution of keratins to cell and tissue integrity, al- The authors declare no conflict of interest. though the underlying pathomechanisms are not yet understood This article is a PNAS Direct Submission. (4). Furthermore, loss of keratin expression, together with down- 1 – – K.S. and A.W.F. contributed equally to this work. regulation of cell cell and cell matrix contacts, represents an 2To whom correspondence may be addressed. E-mail: [email protected] or important feature of the epithelial–mesenchymal transition [email protected]. (EMT) and is thought to contribute to the increased migratory This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and invasive behavior of metastatic tumor cells. Their properties, 1073/pnas.1310493110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310493110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 be significantly reduced by restoring only small amounts of laser power (stretch), the cells start to deform and display creep keratin K5/K14. behavior JðtÞ followed by relaxation after a subsequent stepwise decrease of the laser power. Addressing the variability of biological Results samples, a minimum of 300 cells for each cell line was used for Strongly Increased Softening in Keratin-Free Cells. To investigate analysis. As a measure of the mechanical properties, we analyzed the contribution of keratins to epithelial cell mechanics, we the median creep deformation J (t = 3 s) at the end of cell established a keratinocyte cell line lacking all type II keratins. In stretching. Comparison of WT and KO cells revealed a drastic accordance with the instability of type I keratins in the absence increase of cell deformability of about 60 ± 20% for the KO cells of type II keratin partners, this resulted in cell lines completely (Fig. 1C). At the same time, deformation curves and J (t = 3s) lacking keratin filaments, without compensatory up-regulation of for KO and K5 rescue cells are similar and lie within each other’s other IF proteins (15, 17). To verify the keratin dependence of confidence bounds (Fig. 1 B and C). Due to the low expression the resulting phenotypes, a rescue keratinocytes cell line reex- level of keratin protein of 13% in K5 cells, the similar deformation pressing K5/K14 filaments stably was developed (named K5 curves are to be expected, suggesting that epithelial cell stiffness cells), which expresses 13% of the keratin protein amount of critically depends on the amount of keratin per cell. the WT (15). Thus, it is possible to investigate the overall bio- The strong reliance on whole cell deformability on keratins mechanical contribution of the keratin cytoskeleton in living could easily be caused by changes in the architecture and/or epithelial cells. To probe whole cell mechanical properties, we use amount of other cytoskeletal components. To rule these out, we an automated microfluidic optical stretcher (μOS), a two-beam used immunofluorescence and Western blots to characterize the laser trap that deforms single suspended cells with optically in- various cell lines for keratin, actin, and tubulin. Absence of ker- duced surface forces (Fig. 1A) (16). On a stepwise increase of the atin filaments can clearly be seen in KO keratinocytes, whereas A 5µm laser pattern capillary d(0) 0.8 stretch / W fibers stretch cells P trap relaxation 0.1 objective 0 0 1 2 t / s 3 4 5 B 0.06 WT (396 cells) C 0.06 n.s. KO (336 cells) *** 0.05 K5 (552 cells) 0.05 0.04 0.04 0.03 0.03 0.02 0.02 = 3s)/ arb. units t J(t) / arb. units (552 cells) ( 0.01 (336 cells) J 0.01 laser pattern 0 (396 cells) 0 0 1 2 t / s 3 4 5 WT KO K5 D keratin 5 actin tubulin merge Fig.