Micromechanical properties of keratin intermediate filament networks

Abstract

E pithelial cells form an indispensable physical barrier to withstand mechanical forces in the surrounding environment. They express cell-type-specific keratin intermediate filament (KIF) proteins, which assemble into complex networks of 10-nm-diameter fibers made up of heterodimers of acidic type I and neutral-basic type II isoforms (1). The primary rat alveolar epithelial cells (AECs) used in this study contain polymers of keratin 8 and 18 (K8/K18) (2). In general, intermediate filaments maintain the mechanical integrity of cells (3-5). For example, K8 knockout mutation is embryonic lethal in the mouse because of hemorrhaging in the liver (6), and mutations in epidermal keratins cause skin-blistering diseases (7).The eukaryotic cytoskeleton comprises intermediate filaments (IFs), microfilaments (MFs), and microtubules (MTs). All are dynamic structures, undergoing continuous assembly and disassembly in the intracellular environment (8-10). However, KIF assembly differs significantly from MT and MF (8,9), in that it is nucleotide-independent and that the critical concentration of subunits required for assembly is lower than required for MT and MF. Intermediate filaments resist conditions such as detergent treatment with or without high-salt extraction (11), which disassemble and solubilize the constituent proteins of MT and MF. KIF can be efficiently disassembled only by employing high concentrations of denaturants such as urea (4). We took advantage of the robust nature of the KIF network to measure its mechanical properties, without perturbation by MTs and MFs, in AECs by using particle-tracking microrheology (PTM) (12-14).We show that there is a correlation between the architecture of the KIF network and the local mechanical properties in AECs, and that shear stress applied across the cell surface causes a structural remodeling of KIF and a substantial increase in the elastic modulus of the network. Results and DiscussionShear Stress Alters the Mesh Size of KIF Networks. To understand the specific structural role of the KIF network, we quantified its mechanical properties in the absence of MT and MF. The intact network can be prepared in AECs by detergent treatment and high-salt extraction (4, 10), conditions that remove membranes, soluble cytoplasmic components, and MT and MF (15). To ensure that the extraction procedure did not alter cell shape and size or significantly affect the three-dimensional organization of the KIF network, AECs were transiently transfected with a construct expressing GFP-K18 (10). Cells were imaged pre-and postextraction by confocal microscopy; neither cell height (total depth of the Z-stack) nor the organization of the KIF was altered [supporting information (SI) Fig. 5].The ultrastructure of the KIF network, now devoid of MT and MF (SI Fig. 6), was examined by electron microscopy (11, 15). Cell images were divided into three zones that did not vary significantly across the cell population (SI Fig. 7A). As shown in Fig. 1A, the mesh size within the KIF network, defined as the mean ...

Keratins as the main component for the mechanical integrity of keratinocytes

“…Strain stiffening goes along with a predominantly elastic behavior in biomimetic systems (19). A similar elastic response was also found in particle-tracking microrheology experiments on epithelial cells (20). Furthermore, on the level of transgenic mice and patients with heritable fragility of the epidermis, a clear correlation between molecular integrity of keratin filaments (K5 and K14) and mechanical toughness of epithelia was shown (21,22).…”

“…On the other hand, it has been proposed that IFs serve as a major mechanical buffer system protecting cells from environmental stress (5). Seminal publications have tried to shed light on this hypothesis by use of biomimetic model systems on artificial IF networks (15), extracted keratin networks (20,31), and analyses on whole cells or mouse models (32,33). In essence, many of these analyses identified a very low bending stiffness for keratin filaments and a minor effect of keratin on cell elasticity.…”

Ubiquitin-Proteasome-mediated Degradation of Keratin Intermediate Filaments in Mechanically Stimulated A549 Cells

“…Following formaldehyde fixation, cells were permeabilized with 0.05% Tween 20 for 5 min, washed 3ϫ with PBS, and processed for indirect immunofluorescence as previously described (13,14). Following staining, the glass slides were washed in PBS and mounted in gelvatol containing 100 mg/ml Dabco (1,4-diazabicyclo [2.2.2] octane; Aldrich Chemical (14)).…”

“…Cells were incubated in a humidified atmosphere of 5% CO 2 /95% air at 37°C. An in vitro model for shear stress was used to examine the effect of physical forces on keratin IFs in A549 cells, as previously described (1,13). A549 cells were cultured on 25 ϫ 75 ϫ 1 mm glass slides and subjected to continuous laminar flow to generate shear stress (7.5, 15, and 30 dynes/cm 2 ) using the Flexcell Streamer Device.…”

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

“…Intermediate filaments form the largest group of cytoskeletal proteins (1)(2)(3)(4), and among them keratins constitute the most diverse family. At least 35-37 keratin (K) 3 polypeptides (K1 to K37) are known until now, which are predominantly expressed in human epithelial tissues.…”