Keratins as the main component for the mechanical integrity of keratinocytes Lena Rammsa,1, Gloria Fabrisa,1, Reinhard Windofferb, Nicole Schwarzb, Ronald Springera, Chen Zhouc, Jaroslav Lazarc, Simone Stiefela, Nils Herscha, Uwe Schnakenbergc, Thomas M. Magind, Rudolf E. Leubeb, Rudolf Merkela, and Bernd Hoffmanna,2 aInstitute of Complex Systems, ICS-7: Biomechanics, Forschungszentrum Jülich, 52425 Jülich, Germany; bInstitute of Molecular and Cellular Anatomy, Rheinisch-Westfaelische Technische Hochschule, Aachen University, 52057 Aachen, Germany; cInstitute of Materials in Electrical Engineering 1, RWTH Aachen University, 52074 Aachen, Germany; and dTranslational Centre for Regenerative Medicine and Institute of Biology, University of Leipzig, 04103 Leipzig, Germany Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved October 8, 2013 (received for review July 17, 2013) Keratins are major components of the epithelial cytoskeleton and keratin subunits (14), the total and isotype-specific contribution are believed to play a vital role for mechanical integrity at the of the overall keratin network to mechanical properties of epi- cellular and tissue level. Keratinocytes as the main cell type of the thelia remains highly challenging to analyze in vivo. An additional epidermis express a differentiation-specific set of type I and type II difficulty in experimentation is the absence of drugs to specifically keratins forming a stable network and are major contributors of disrupt the keratin IF system. keratinocyte mechanical properties. However, owing to compen- Most results on the mechanical properties and functions of IFs satory keratin expression, the overall contribution of keratins to are based on biomimetic systems and disease models. One of the cell mechanics was difficult to examine in vivo on deletion of single hallmarks of IFs is their low bending stiffness. In conjunction keratin genes. To overcome this problem, we used keratinocytes with nonlinear strain stiffening observed in networks at large lacking all keratins. The mechanical properties of these cells were deformations, these features supposedly enable IFs to serve as a mechanical buffer system protecting cells from environmental analyzed by atomic force microscopy (AFM) and magnetic tweezers – experiments. We found a strong and highly significant softening of stress (15 17). High tensional loads have already been observed keratin-deficient keratinocytes when analyzed by AFM on the cell for keratins in vivo (18). Strain stiffening goes along with a pre- dominantly elastic behavior in biomimetic systems (19). A similar body and above the nucleus. Magnetic tweezers experiments fully elastic response was also found in particle-tracking microrheology confirmed these results showing, in addition, high viscous contri- experiments on epithelial cells (20). Furthermore, on the level of butions to magnetic bead displacement in keratin-lacking cells. transgenic mice and patients with heritable fragility of the epi- Keratin loss neither affected actin or microtubule networks nor dermis, a clear correlation between molecular integrity of keratin their overall protein concentration. Furthermore, depolymerization filaments (K5 and K14) and mechanical toughness of epithelia was of actin preserves cell softening in the absence of keratin. On shown (21, 22). reexpression of the sole basal epidermal keratin pair K5/14, the Generation of keratin KO mouse strains by deleting either the keratin filament network was reestablished, and mechanical type I or type II keratin gene cluster (23) results in complete loss properties were restored almost to WT levels in both experimental of keratin filaments in epidermal cells and provides the unique setups. The data presented here demonstrate the importance of opportunity to accomplish accurate and exact biomechanical keratin filaments for mechanical resilience of keratinocytes and analyses of keratin-free keratinocytes for the very first time. indicate that expression of a single keratin pair is sufficient for Until now, these cell systems have been characterized for various almost complete reconstitution of their mechanical properties. Significance ormation of a barrier capable of protecting tissue from ex- Fternal damage, chemical factors, and pathogens while resist- For decades, researchers have been trying to unravel one of the ing mechanical stress, external pressure, or shear force is one of key questions in cell biology regarding keratin intermediate fil- the main functions of epithelial tissues. Keratinocytes represent ament function in protecting epithelial cells against mechanical the major cell type of mammalian epidermis and are mainly stress. For many different reasons, however, this fundamental responsible for barrier functionality (1, 2). On the molecular hypothesis was still unproven. Here we answer this pivotal level, mechanical cell properties mainly depend on cytoskeletal question by the use of keratin KO cells lacking complete keratin fibrous structures (3), namely actin filaments, microtubules, and gene clusters to result in total loss of keratin filaments. This lack intermediate filaments (IFs). Although the contribution of actin significantly softens cells, reduces cell viscosity, and elevates filaments and microtubules to the resilience of many cell types is plastic cell deformation on force application. Reexpression of widely accepted (4), it has been hypothesized for many years that single keratin genes facilitates biomechanical complementation the resilience of epithelia against various types of deformation of complete cluster loss. Our manuscript therefore makes a very depends largely on keratins (5–7). These form a stable network strong case for the crucial contribution of keratins to cell me- spanning from the cell periphery to the nucleus. Peripheral fila- chanics, with far-reaching implications for epithelial patho- ments dynamically enlarge into thicker filaments and progressively physiology. intermingle with the preexisting network by continuously moving centripetally until, in the center of the cell, a dense network of Author contributions: R.W., U.S., T.M.M., R.E.L., R.M., and B.H. designed research; L.R., G.F., BIOPHYSICS AND keratin filaments encircles the nucleus (8, 9). R.W., N.S., C.Z., J.L., S.S., and N.H. performed research; C.Z., J.L., U.S., and T.M.M. contrib- COMPUTATIONAL BIOLOGY Keratins are encoded by a large multigene family of more than uted new reagents/analytic tools; L.R., G.F., R.W., R.S., and R.M. analyzed data; and R.E.L., R.M., and B.H. wrote the paper. 50 genes that are specifically expressed depending on distinct fl developmental pathways and physiological requirements (10, 11). The authors declare no con ict of interest. Based on their amino acid sequence, type I keratins display an This article is a PNAS Direct Submission. overall acidic character and differ considerably from the more 1L.R. and G.F. contributed equally to this work. basic type II keratins (12). Filament assembly requires both ker- 2To whom correspondence should be addressed. E-mail: [email protected]. PHYSICS atin types because of the obligatory heterodimer composition of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. keratin IFs (13). Given that most epithelia express 4–10 different 1073/pnas.1313491110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1313491110 PNAS | November 12, 2013 | vol. 110 | no. 46 | 18513–18518 Downloaded by guest on September 29, 2021 cell biological and biochemical functions (24, 25). Of note, in- creased cell motility and altered desmosomal and hemidesmosomal adhesion were identified in these studies. In the current work, we analyze the keratin type I and type II mutant keratinocyte cell lines and compare them to WT controls established in parallel and rescue cell lines reexpressing keratins 5 and 14. Mechanical properties are examined in living cells from outside by atomic force microscopy (AFM) (26, 27) and from inside by magnetic tweezers (28) (Fig. S1). The data provide compelling evidence for the hypothesis that keratins determine the mechanical stability and integrity of epithelial cells by direct effects on various mechanical parameters such as elasticity, vis- cosity, spring constant, and relaxation time. Results Loss of Keratin Filaments Leads to Significant Cell Softening. Keratin filaments are believed to strengthen epithelial cells against various types of mechanical stress, mainly indentation, local deformation, and strain. To investigate this hypothesis, we characterized the mechanical properties of WT and keratin-lacking cell lines by complementary approaches, i.e., by AFM and magnetic tweezers experiments. To consolidate our data beyond doubt, both keratin − − − − type I / mutant cells (KtyI / ) and keratin type II mutant cells − − (KtyII / ), as well as corresponding rescue cell lines, were exam- ined in parallel. − − In a first series of experiments, WT and KtyI / cell lines were Fig. 2. AFM force-distance curves and force-indentation plots. (A) Plot showing the superposition of three AFM force-distance curves (green, red, characterized for their resilience against external force by AFM blue) recorded successively at 5-s intervals on the same position above the indentation. Immunocytochemistry stainings revealed well- fi nucleus of a WT keratinocyte. (B) Plot showing the average forces needed to developed keratin networks in WT cells with thin keratin la- reach a certain indentation depth above the nucleus (n = 109–125, ments