Structural Hierarchy of Trichocyte Keratin Intermediate Filaments.

Although trichocyte keratins (hair, wool, quill, claw) have been studied since the 1930s it is only over the last 30 years or so that major advances have been made in our understanding of the complex structural hierarchy of the filamentous component of this important filament-matrix composite. A variety of techniques, including amino acid sequence analysis, computer modelling, X-ray fibre diffraction and protein crystallography, various forms of electron microscopy, and crosslinking methods have now combined to reveal much of the structural detail. The heterodimeric structure of the keratin molecule is clear, as are the highly-specific modes by which these molecules aggregate to form functionally viable IF. The observation that hair keratin can adopt not one but two structurally-distinct conformations, one formed in the living cells at the base of the hair follicle in a reducing environment and the second in the fully differentiated hair in dead cells in an oxidized state, was unexpected but has major implications for the mechanism of hair growth. Insights have also been made into the mechanism of the uppermost level of hair superstructure, relating to the assembly of the IF in the paracortical and orthocortical macrofibrils.

Intermediate filament structure in fully differentiated (oxidised) trichocyte keratin.

For the past 50years there has been considerable debate over the sub-structure of the fully differentiated (oxidised) trichocyte keratin intermediate filament. Depending on the staining and preparative procedures employed, IF observed in transverse section in the transmission electron microscope have varied in appearance between that of a "ring" and a "ring-core" structure, corresponding to the so-called (8+0) and (7+1) protofilament arrangements. In a new analysis of the fine structure of the 1nm equatorial region of the X-ray diffraction pattern of quill we show that the observed pattern is consistent with the (8+0) model and we are also able to assign values to the various parameters. In contrast, we show that the observed X-ray pattern is inconsistent with a (7+1) arrangement. Furthermore, in the (7+1) model steric hindrance would be encountered between the core protofilament and those constituting the ring. The appearance of a central "core" in transverse TEM sections, previously attributed to a central protofilament, is explained in terms of portions of the apolar, disulfide-bonded head and/or tail domains of the trichocyte keratin IF molecules, including the conserved H subdomains, lying along the axis of the IF, thereby decreasing the efficacy of the reducing agents used prior to staining. The H1 subdomain, previously shown to be important in the assembly of epidermal IF molecules at the two- to four-molecule level, is likely to have a similar role for the trichocyte keratins and may form part of a central scaffold on which the molecules assemble into fully functional IF.

Structural Transition of Trichocyte Keratin Intermediate Filaments During Development in the Hair Follicle.

The intermediate filaments (IF) in trichocyte (hard α-) keratin are unique amongst the various classes of IF in having not one but two topologically-distinct structures. The first is formed at an early stage of hair development in a reducing environment within the cells in the lower part of the follicle. The second structure occurs at a later stage of hair development in the upper part of the follicle, where there is a transition to an oxidizing environment. Crosslinking studies reveal that molecular slippage occurs within the IF upon oxidation and that this results in many cysteine residues lying in near axial alignment, thereby facilitating disulphide bond formation. The disulphide bonds so formed stabilize the assembly of IF molecules and convert the keratin fibre into a tough, resilient and insoluble structure suitable for its function in vivo as a thermo-regulator and a protector of the animal against its external environment.

Ultrastructural localization of hair keratins, high sulfur keratin-associated proteins and sulfhydryl oxidase in the human hair.

Hardening of the human hair shaft during cornification results from the bonding of keratins and keratin-associated proteins. In situ hybridization and light immunocytochemical studies have shown the general distribution of different keratins and some associated proteins but not determined their ultrastructural localization. I report here the localization of hair keratins, two high-sulfur keratin-associated proteins and sulfhydryl oxidase has been studied under the transmission electron microscope in the cornification zone of the human hair. The ultrastructural study on keratin distribution in general confirms previous light microscopic studies. Sulfur-rich KAP1 is mainly cortical but the labeling disappears in fully cornified cortical cells while a diffuse labeling is also present in differentiating cuticle cells. Sulfur-rich K26 immunolocalization is only detected in the exocuticle and endocuticle. Sparse labeling for sulfhydryl oxidase occurs in differentiating cortical cells but is weak and uneven in cuticle cells and absent in medulla and inner root sheath. Labeling disappears in the upper fully cornified cortex and cuticle. The observations indicate that sulfhydryl oxidase and keratin associated proteins are initially produced in the cytoplasm among keratin bundles accumulating in cortical and cuticle cells but these proteins undergo changes during the following cornification that alter the epitopes tagged by the antibodies.

Culturing fibroblasts in 3D human hair keratin hydrogels.

Human hair keratins are readily available, easy to extract, and eco-friendly materials with natural bioactivities. Keratin-based materials have been studied for applications such as cell culture substrates, internal hemostats for liver injury, and conduits for peripheral nerve repair. However, there are limited reports of using keratin-based 3D scaffolds for cell culture in vitro. Here, we describe the development of a 3D hair keratin hydrogel, which allows for living cell encapsulation under near physiological conditions. The convenience of making the hydrogels from keratin solutions in a simple and controllable manner is demonstrated, giving rise to constructs with tunable physical properties. This keratin hydrogel is comparable to collagen hydrogels in supporting the viability and proliferation of L929 murine fibroblasts. Notably, the keratin hydrogels contract less significantly as compared to the collagen hydrogels, over a 16-day culture period. In addition, preliminary in vivo studies in immunocompetent animals show mild acute host tissue response. These results collectively demonstrate the potential of cell-loaded keratin hydrogels as 3D cell culture systems, which may be developed for clinically relevant applications.

Calcification provides mechanical reinforcement to whale baleen alpha-keratin.

Hard alpha-keratins such as hair, nail, wool and horn are stiff epidermal appendages used by mammals in a variety of functions including thermoregulation, feeding and intraspecific competition. Hard alpha-keratins are fibre-reinforced structures consisting of cytoskeletal elements known as 'intermediate filaments' embedded in an amorphous protein matrix. Recent research has shown that intermediate filaments are soft and extensible in living keratinocytes but become far stiffer and less extensible in keratinized cells, and this stiffening may be mediated by air-drying. Baleen, the keratinous plates used by baleen whales during filter feeding, is an unusual mammalian keratin in that it never air dries, and in some species, it represents the most heavily calcified of all the hard alpha-keratins. We therefore tested the hypothesis that whale baleen is stiffened by calcification. Here, we provide, to our knowledge, the first comprehensive description of baleen material properties and show that calcification contributes to overcoming the shortcomings of stiffening this hard alpha-keratin without the benefit of air-drying. We also demonstrate striking interspecies differences in the calcification patterns among three species of baleen whales and provide novel insights into the function and evolution of this unusual biomaterial.

Cortical cell types and intermediate filament arrangements correlate with fiber curvature in Japanese human hair.

Naturally straight and curved human scalp hairs were examined using fluorescence and electron microscopy techniques to determine morphological and ultrastructural features contributing to single fiber curvature. The study excluded cuticle and medulla, which lack known bilateral structural asymmetry and therefore potential to form curved fibers. The cortex contained four classifiable cell types, two of which were always present in much greater abundance than the remaining two types. In straight hair, these cell types were arranged annularly and evenly within the cortex, implying that the averaging of differing structural features would maintain a straight fiber conformation. In curved fibers, the cell types were bilaterally distributed approximately perpendicular to fiber curvature direction with one dominant cell type predominantly located closest to the convex fiber side and the other, closest to the concave side. Electron tomography confirmed that the dominant cell type closest to the convex fiber side contained discrete macrofibrils composed of helically arranged intermediate filaments, while the dominant cell type closest to the concave side contained larger fused macrofibrils composed of intermediate filament arrangements varying from helical to hexagonal arrays approximately parallel to the longitudinal fiber axis. These findings concur with the current hypothesis of hair curvature formation and behavior.