These observations suggested that soluble keratin protein subunits exist in equilibrium with filamentous (polymerized) protein, a hypothesis since confirmed by experiments on living cells expressing GFP-keratin fusion proteins (14, 15).
Small amounts of soluble keratins (∼5%) have also been identified in some cells (11, 12, 13).
Newly expressed mutant keratins (8) or tissue-derived keratins microinjected into epithelial cells (9, 10) readily integrate into preexisting keratin networks. The once commonly held view of the keratin cytoskeleton as a relatively inert entity has been overturned in light of recent experiments showing that KIFs exhibit a remarkable range of dynamic properties, at both molecular and supramolecular levels. However, there is little evidence for a mechanical role for keratins in simple epithelia, comparable to that of the keratins in stratified epithelia. Although the functions of the keratins of simple epithelia are less well understood, they have been implicated in many other cellular processes, including cell signaling, apoptosis, cell cycle progression, gene transcription, and cytoprotection (4, 7).
2110 ADOBE DRIVE IL SKIN
This important role was highlighted by the discovery that mutations in the highly conserved, central α-helical rod domains of K5/14 or K1/10 cause a variety of human skin fragility diseases (5, 6). One of the major functions of KIFs in stratified epithelia is to enable cells to withstand mechanical stresses, for example, frictional forces or strain (3). Simple epithelial cells, found in liver, kidney, pancreas, intestine, and lung, all express the highly conserved keratins 8 and 18 (K8 and K18), often in association with variable amounts of secondary keratins, including keratins 7, 19, 20, and 23 (4). Different combinations of Type I and Type II proteins, known as keratin “pairs,” are coordinately expressed in epithelial cells during development (3). Keratin IFs (KIFs) are expressed in both simple and stratified epithelia as obligate heteropolymers, assembled from at least one Type I and one Type II protein (2). Type I (acidic) and Type II (neutral-basic) keratins constitute the largest group of intermediate filament (IF) proteins in the cytoskeleton of eukaryotic cells (1). Insights into the mechanical properties of epithelial cells: the effects of shear stress on the assembly and remodeling of keratin intermediate filaments. We suggest that keratin particles constitute a reservoir of protein that can be recruited into KIFs under flow, creating a more robust cytoskeleton able to withstand shear forces more effectively.-Flitney, E. The particles that remained after shearing were phosphorylated and were closely associated with KIFs. Both effects were accompanied by the disappearance of most keratin particles and by increased phosphorylation of K8 and K18 on serine residues 73 and 33, respectively. Shear stress dramatically reduced the soluble keratin component and transformed the fine bundles of KIFs into thicker, “wavy” tonofibrils. Triton X-100 extracted ∼10% of the cellular keratin, and this was accompanied by a loss of the particles but not the KIFs. Under normal culture conditions, immunofluorescence revealed a delicate network of fine tonofibrils containing KIFs, together with many nonfilamentous, keratin-containing “particles,” mostly containing either keratin 8 (K8) or 18 (K18), but not both. The effects of shear stress on the keratin intermediate filament (KIF) cytoskeleton of cultured human alveolar epithelial (A549) cells have been investigated.
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