Types I and II Keratin Intermediate Filaments

Abstract

Keratins-types I and II-are the intermediate-filament-forming proteins expressed in epithelial cells. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type-, differentiation-, and context-dependent manner. Here, we review how keratins serve multiple homeostatic and stress-triggered mechanical and nonmechanical functions, including maintenance of cellular integrity, regulation of cell growth and migration, and protection from apoptosis. These functions are tightly regulated by posttranslational modifications and keratin-associated proteins. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility or altered tissue homeostasis. Furthermore, keratin mutation or misregulation represents risk factors or genetic modifiers for several additional acute and chronic diseases.

Figure 1.

Introduction to keratin intermediate filaments (IFs). (A) Schematic representation of the tripartite domain structure shared by all keratin and other IF proteins. A central α-helical “rod” domain acts as the major determinant of self-assembly and is flanked by nonhelical “head” and “tail” domains at the amino and carboxyl termini, respectively. Within this 310-amino-acid-long rod domain, the heptad-repeat-containing segments (e.g., coils 1A, 1B, and 2) are interrupted by linker sequences at two conserved locations (e.g., L1 and L12). Rod domain boundaries consist of highly conserved 15–20-amino-acid regions (shown in orange) that are crucial for polymerization and frequently mutated in human disease (see www.interfil.org). (B) Visualization of filaments, reconstituted in vitro from purified human K5 and K14 proteins, by negative staining and electron microscopy. Scale bar, 100 nm. (C) Visualization of native keratin IFs in the stratum corneum layer of human epidermis using cryo-transmission electron microscopy on a fully hydrated, vitreous tissue specimen (Norlén and Al-Amoudi 2004). Bundles of tightly packed keratin IFs run parallel (para) or perpendicular (perp) to the sectioning plane. Scale bar, 50 nm. (Inset) Detailed views of filaments in cross section, shown at two magnifications. As many as seven subfibrils, including a centrally located one, can be seen. Scale bar, 20 nm. (D) Skin epidermal keratinocytes in culture. Dual-immunofluorescence labeling for keratin (red signal) and desmoplakin (green signal), a desmosome component. Keratin IFs are organized in a network that spans the whole cytoplasm and are attached to desmosomes, which are points of adhesion at cell–cell contacts. n, Nucleus. Scale bar, 20 µm. (E) Gut epithelial wall in cross section, emphasizing the epithelium. This fresh-frozen specimen was triple-labeled for K8/K18 (red signal), K19 (green signal), and nuclei (blue signal). Note the concentration of staining at the apical pole of enterocytes. The star denotes a goblet cell, which also features a prominent K8/K18 network in the cytoplasm. bl, Basal lamina; lum, lumen; n, nucleus. Scale bar, 20 µm. (Reprinted, with permission, from Kim and Coulombe 2007; C, originally adapted from Norlén and Al-Amoudi 2004, with permission from Elsevier.)

Figure 2.

Phylogenic tree of human keratins. (A) Comparison of the primary structure of human keratins using the publicly available ClustalW and TreeView softwares. Sequence relatedness is inversely correlated with the length of the lines connecting the various sequences, as well as the number and position of branchpoints. This comparison makes use of the sequences from the head and central rod domain for each keratin. Two major branches are seen in this tree, corresponding exactly to the known partitioning of keratin genes into types I and II sequences. Beyond this dichotomy, each subtype is further segregated into major subgroupings. (B) Location and organization of genes encoding types I and II keratins in the human genome. All functional type I keratin genes, except Krt18, are clustered on the long arm (q) of human chromosome 17, whereas all functional type II keratin genes are located on the long arm of chromosome 12. Krt18, a type I gene, is located at the telomeric (Tel) boundary of the type II gene cluster. The suffix P identifies keratin pseudogenes. As highlighted by the color code used in frames A and B, individual type I and type II keratin genes belonging to the same subgroup, based on the primary structure of their protein products, tend to be clustered in the genome. Moreover, highly homologous keratin proteins (e.g., K5 and K6 paralogs; also, K14, K16, and K17) are often encoded by neighboring genes, pointing to the key role of gene duplication in generating keratin diversity. These features of the keratin family are virtually identical in mouse (not shown). (Adapted from Coulombe et al. 2013.)

Figure 3.

Loss of keratin K14 elicits epidermolysis bullosa simplex (EBS)-like features in mouse skin. (A) Leg skin in a patient suffering from the Dowling–Meara (severe) form of EBS. Characteristic of this severe variant of this disease, several skin blisters are grouped in a herpetiform fashion. (B,C) Images of newborn mouse littermates, comparing K14-null and wild-type (WT) mice. The K14-null neonate shows massive skin blistering. The front paws and facial area are severely affected (arrows); by comparison, a WT littermate shows intact skin. (D,E) Micrographs from hematoxylin/eosin-stained histological sections prepared from the front paws of 2-d-old K14-null and WT mice. Epidermal cleavage is obvious in the K14-null sample. Loss of epidermal integrity occurs through the basal layer of keratinocytes (labelled “blister”)—the defining characteristic of EBS. Three basal keratinocytes are boxed in E. epi, epidermis; hf, hair follicle. Scale bar, 100 µm. (A, Reprinted from Coulombe et al. 2013; B–E, adapted from Coulombe et al. 2009.)

Figure 4.

Posttranslational modifications (PTMs) of intermediate filament (IF) proteins. (A) IFs undergo multiple PTMs, including phosphorylation (PO₄), O-linked glycosylation (GlcNAc), ubiquitination (Ub), acetylation (acetyl), SUMOylation (SUMO), transamidation, and farnesylation (Snider and Omary 2014). Keratins also undergo all these modifications except for farnesylation, which specifically occurs in lamin IF proteins. Multiple functions are ascribed to IF/keratin PTMs, as highlighted in the panel text boxes. (B) Keratin PTMs target residues that can be modified by multiple PTMs. For example, Ser/Thr residues can be phosphorylated and glycosylated, whereas Lys residues can be modified by acetylation, ubiquitination, SUMOylation, and transamidation. There is also cross talk between different modifications, as exemplified by the effect of phosphorylation on several PTMs, and vice versa. The multiple combinations of modifications on a given keratin can provide a complex hierarchy of regulation. (Modified from Snider and Omary 2014.)