The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress.

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central "rod" region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.

Age-associated thin hair displays molecular, structural and mechanical characteristic changes.

Hair thinning occurs during normal chronological aging in women and in men leading to an increased level of thinner hair shafts alongside original thicker shafts. However, the characteristics of age-associated thin hairs remain largely unknown. Here we analyzed these characteristics by comparing at multiscale thin and thick hairs originated from Caucasian women older than 50 years. We observed that the cortex of thick hair contains many K35(+)/K38(-) keratinocytes that decrease in number with decreasing hair diameter. Accordingly, X-ray diffraction revealed differences supporting that thin and thick hairs are different with regards to the nature of the intermediate filaments making up their cortices. In addition, we observed a direct correlation between hair ellipticity and diameter with thin hairs having an unexpected round shape compared to the elliptic shape of thick hairs. We also observed fewer cuticle layers and a reduced frequency of a medullae in thin hairs. Regarding mechanical properties, thin hairs exhibited a surprising increased rigidity, a decrease of the viscosity and a decrease of the water diffusion coefficient. Hence, aged-associated thin hairs exhibit numerous modifications likely due to changes of hair differentiation program as evidenced by the modulations in the expression of hair keratins and keratin-associated proteins and by the X-ray diffraction specters. Hence, hair thinning with age does not consist simply of the production of a smaller hair. It is rather a more profound process likely relying on the implementation of an "aged hair program" that takes place within the hair follicle.

Wool fiber curvature is correlated with abundance of K38 and specific keratin-associated proteins.

Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.

Processing hair follicles for transmission electron microscopy.

Transmission electron microscopy (TEM) has greatly advanced our knowledge of hair growth and follicle morphogenesis, but complex preparations such as fixation, dehydration and embedding compromise ultrastructure. While recent developments with cryofixation have been shown to preserve the ultrastructure of biological materials close to native state, they do have limitations. This review will focus on each stage of the TEM sample preparation process and their effects on the structural integrity of follicles.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

Insights in Human Hair Curvature by Proteome Analysis of Two Distinct Hair Shapes.

Scalp hair is a universal human characteristic, and a wide range of hair shape and color variations exists. Although differences in human scalp hair shape are visually apparent, the underpinning molecular insights are yet to be fully explored. This work reports the determination of differences at the protein level between two distinct groups of hair shape: very straight samples versus very curly hair samples. An in-depth highresolution liquid-chromatography mass spectrometry proteome analysis study was performed on hair samples from 50 individuals (pooled in 10 × 5 samples) with very curly hair and 50 subjects with very straight hair (pooled in 10 × 5 samples) to decipher differences between the two experimental groups at the protein level. Our results demonstrate that a distinction between the two experimental groups (very straight vs. very curly) can be made based on their overall protein profiles in a multivariate analysis approach. Further investigation of the protein expression levels between these two groups pinpointed 13 unique proteins which were found to be significantly different between the two groups, with an adjusted p-value < 0.05 and a fold change of more than two. Although differences between the very curly and the very straight hair sample groups could be identified, linkage between population differences and curl phenotype is currently unknown and requires further investigation.

Helical twist direction in the macrofibrils of keratin fibres is left handed.

Macrofibrils, the main structural features within the cortical cells of mammalian hair shafts, are long composite bundles of keratin intermediate filaments (KIFs) embedded in a matrix of keratin-associated proteins. The KIFs can be helically arranged around the macrofibril central axis, making a cylinder within which KIF helical angle relative to macrofibril axis increases approximately linearly from macrofibril centre to edge. Mesophase-based self-assembly has been implicated in the early formation of macrofibrils, which first appear as liquid-crystal tactoids in the bulb of hair follicles. Formation appears to be driven initially by interactions between pre-keratinized KIFs. Differences in the nature of these KIF-KIF interactions could result in all macrofibrils being internally twisted in a single handedness, or a 50:50 mixture of handedness within each cortical cell. We data-mined 41 electron tomograms containing three-dimensional macrofibril data from previously published studies of hair and wool. In all 644 macrofibrils examined we found that within each tomogram all macrofibrils had the same handedness. We concluded that earlier reports of left- and right-handed macrofibrils were due to artefacts of imaging or data processing. A handedness marker was used to confirm (using re-imaged sections from earlier studies) that, in both human and sheep, all macrofibrils are left-handed around the macrofibril axis. We conclude that this state is universal within mammalian hair. This also supports the conclusion that the origin of macrofibril twist is the expression of chiral twisting forces between adjacent KIFs, rather than mesophase splay and bending forces relaxing to twisting forces acting within a confined space.

Wanted, dead and alive: Why a multidisciplinary approach is needed to unlock hair treatment potential.

Human recorded history is littered with attempts to improve the perceived appearance of scalp hair. Throughout history, treatments have included both biological and chemical interventions. Hair "quality" or "perceived appearance" is regulated by multiple biological intervention opportunities: adding more hairs by flipping follicles from telogen to anagen, or delaying anagen follicles transiting into catagen; altering hair "apparent amount" by modulating shaft diameter or shape; or, in principle, altering shaft physical properties changing its synthesis. By far the most common biological intervention strategy today is to increase the number of hairs, but to date this has proven difficult and has yielded minimal benefits. Chemical intervention primarily consists of active material surface deposition to improve shaft shine, fibre-fibre interactions and strength. Real, perceptible benefits will best be achieved by combining opportunity areas across the three primary sciences: biology, chemistry and physics. Shaft biogenesis begins with biology: proliferation in the germinative matrix, then crossing "Auber's Critical Line" and ceasing proliferation to synthesize shaft components. Biogenesis then shifts to oxidative chemistry, where previously synthesized components are organized and cross-linked into a shaft. We herein term the crossing point from biology to chemistry as "The Orwin Threshold." Historically, hair biology and chemistry have been conducted in different fields, with biological manipulation residing in biomedical communities and hair shaft chemistry and physics within the consumer care industry, with minimal cross-fertilization. Detailed understanding of hair shaft biogenesis should enable identification of factors necessary for optimum hair shaft production and new intervention opportunities.

Expression and purification of high sulfur and high glycine-tyrosine keratin-associated proteins (KAPs) for biochemical and biophysical characterization.

Keratin-associated proteins (KAPs) were identified 70 years ago in wool follicles. KAPs are encoded by several multi-gene families and are classified into three different groups: ultra-high sulfur (UHS), high sulfur (HS) and high glycine-tyrosine (HGT). KAPs are the major constituent of the matrix between the hair keratin intermediate filaments (IFs), and stabilise hair structure by extensive disulfide bonding. However, detailed molecular structural information is lacking for KAPs and for KAP interactions with IFs. As a preliminary step towards their biophysical and structural characterization, we have expressed and purified a HS KAP (KAP11.1) and a HGT KAP (KAP6.1). The expression and purification of KAPs is challenging because they are cysteine-rich proteins with unusual amino acid compositions, they tend to be insoluble in isolation and are prone to forming aggregates in solution. Here we describe the high yield production of pure, soluble KAPs in a chaotrope- and detergent-free buffer. This method has the potential to be used for the overproduction of other KAPs and similar cysteine-rich proteins with high isoelectric points.

Intrinsic curvature in wool fibres is determined by the relative length of orthocortical and paracortical cells.

Hair curvature underpins structural diversity and function in mammalian coats, but what causes curl in keratin hair fibres? To obtain structural data to determine one aspect of this question, we used confocal microscopy to provide in situ measurements of the two cell types that make up the cortex of merino wool fibres, which was chosen as a well-characterised model system representative of narrow diameter hairs, such as underhairs. We measured orthocortical and paracortical cross-sectional areas, and cortical cell lengths, within individual fibre snippets of defined uniplanar curvature. This allowed a direct test of two long-standing theories of the mechanism of curvature in hairs. We found evidence contradicting the theory that curvature results from there being more cells on the side of the fibre closest to the outside, or convex edge, of curvature. In all cases, the orthocortical cells close to the outside of curvature were longer than paracortical cells close to the inside of the curvature, which supports the theory that curvature is underpinned by differences in cell type length. However, the latter theory also implies that, for all fibres, curvature should correlate with the proportions of orthocortical and paracortical cells, and we found no evidence for this. In merino wool, it appears that the absolute length of cells of each type and proportion of cells varies from fibre to fibre, and only the difference between the length of the two cell types is important. Implications for curvature in higher diameter hairs, such as guard hairs and those on the human scalp, are discussed.

Introduction to Hair Development.

The anagen phase of the hair follicle cycle is when the follicle is configured to grow hair. In short hairs (e.g., mouse underhairs and human eye lashes) anagen phase is short, but in the wool of sheep and in human scalp hair anagen is a prolonged state lasting for years. In this chapter we describe the morphological and biological divisions within the anagen follicle.

Environment of the Anagen Follicle.

Hair follicles are part of the skin. Almost universally, follicles are described as an epithelium-derived tubular down growth into the skin's dermis. Because follicles are complex structures, especially when in anagen phase and configured to actively grow fibres, it is easy to forget that they are part of a crowded environment within the skin. This chapter introduces some of the structures which surround the follicle as well as some of the peripheral parts of the follicle, including follicle groups, and the dermal sheath, vasculature, adipocytes, nerves and the arrector pili muscle.

Fibre Ultrastructure.

Mammalian hair fibres can be structurally divided into three main components: a cuticle, cortex and sometimes a medulla. The cuticle consists of a thin layer of overlapping cells on the surface of the fibre, constituting around 10% of the total fibre weight. The cortex makes up the remaining 86-90% and is made up of axially aligned spindle-shaped cells of which three major types have been recognised in wool: ortho, meso and para. Cortical cells are packed full of macrofibril bundles, which are a composite of aligned intermediate filaments embedded in an amorphous matrix. The spacing and three-dimensional arrangement of the intermediate filaments vary with cell type. The medulla consists of a continuous or discontinuous column of horizontal spaces in the centre of the cortex that becomes more prevalent as the fibre diameter increases.

The Follicle Cycle in Brief.

This chapter presents a very succinct overview of the cyclic biology of the hair follicle as it transitions from the quiescent telogen stage to the anagen stage in which hairs are actively produced before regressing through the catagen stage to telogen.

Macrofibril Formation.

Macrofibrils are the main structural component of the hair cortex, and are a composite material in which trichokeratin intermediate filaments (IFs) are arranged as organised arrays embedded in a matrix composed of keratin-associated proteins (KAPs) and keratin head groups. Various architecture of macrofibrils is possible, with many having a central core around which IFs are helically arranged, an organisation most accurately described as a double-twist arrangement. In this chapter we describe the architecture of macrofibrils and then cover their formation, with most of the material focusing on the theory that the initial stages of macrofibril formation are as liquid crystals.

Development of Hair Fibres.

The growth of hairs occurs during the anagen phase of the follicle cycle. Hair growth begins with basement membrane-bound stem cells (mother cells) around the dermal papilla neck which continuously bud off daughter cells which further divide as a transient amplifying population. Division ceases as cell line differentiation begins, which entails changes in cell junctions, cell shape and position, and cell-line specific cytoplasmic expression of keratin and trichohyalin. As the differentiating cells migrate up the bulb, nuclear function ceases in cortex, cuticle and inner root sheath (IRS) layers. Past the top of the bulb, cell shape/position changes cease, and there is a period of keratin and keratin-associated protein (KAP) synthesis in fibre cell lines, with increases, in particular of KAP species. A gradual keratinization process begins in the cortex at this point and then non-keratin cell components are increasingly broken down. Terminal cornification, or hardening, is associated with water loss and precipitation of keratin. In the upper follicle, the hair, now in its mature form, detaches from the IRS, which is then extracted of material and becomes fragmented to release the fibre. Finally, the sebaceous and sudoriferous (if present) glands coat the fibre in lipid-rich material and the fibre emerges from the skin. This chapter follows the origin of the hair growth in the lower bulb and traces the development of the various cell lines.

Hair Shaft Formation and Mitochondrial Bioenergetics: Combining Biology, Chemistry, and Physics.

Research into biological manipulation of hair "quality" has ebbed and waned but today is in a resurgence. Hair appearance is regulated by multiple intervention opportunities-adding more hairs; increasing hair "amount" by modulating shaft diameter or shape; or, in principle, by altering shaft physical properties by changing its synthesis. It is likely that improved benefits may be achieved by combining multiple areas-minimizing follicle loss and miniaturization, maximizing shaft production, and treating the existing shaft. A previously overlooked opportunity is follicle metabolism: building "better" hairs. Hair production is energy intensive, and it is known that follicle metabolism influences shaft diameter. Multiphoton microscopy enables metabolic investigation of live, growing, human, hair follicles. This allows definition of multiple "zones" with vastly different metabolism: proliferation-where keratinocytes proliferate and migrate into specialized layers; production-proliferation ceases, and synthesis and patterning begin; construction and elongation-the structural framework is seeded and cells extend to create the nascent fiber; and maturation-gradual hardening and transformation into mature shaft. Recent investigations into the transition from construction to maturation reinforce this as a key developmental threshold, where shaft production transforms from a biologically driven into a biochemically driven process. We now name this "Orwin's transition."

The structure of the "amorphous" matrix of keratins.

Various keratin fibers, particularly human hairs, were investigated by transmission electron microscopy, TEM, solid-state 1H NMR and Transient Electro-Thermal Technique, TET. The results converge to suggest that the matrix of keratin fiber cortex, far from being amorphous, has a well-defined nano-scale grainy structure, the size of these grains being around 2-4nm. The size of the grains appears to strongly depend on the chemical treatment of the fiber, on the temperature and on the relative humidity of the environment, as well as on the physiological factors at the level of fiber production in follicle. By suggesting an organization at the nano-scale of the protein chains in these grains, likely to be Keratin Associated Proteins, the results challenge the view of matrix as a homogeneous glassy material. Moreover, they indicate the potential of further investigating the purpose of this structure that appears to reflect not only chemical treatments of keratins but also biological processes at the level of the follicle.

Preparation of wool follicles for proteomic studies.

A variety of techniques were applied to wool follicles stored in William's E culture medium to optimise the extraction of keratin and keratin associated proteins (KAPs). A time course study indicated that the maximum storage time for live skin in this buffer at 20 °C was 24 h, after which degradative loss of protein became significant. Maceration of the skin for 10 min followed by reciprocal action shaking for 14 h had a detrimental effect on keratin extractability. The best approach involved using a Dounce homogeniser as this resulted in the highest amount of Type I and II keratins and KAPs.

The proteomics of wool fibre morphogenesis.

Gel and gel-free proteomic techniques have been used for the first time to directly study the proteins present in whole wool follicles and dissected portions of follicles that correlated with morphological changes in the developing fibre as determined by transmission electron microscopy. Individual wool follicles were dissected into four portions designated as the bulb, elongation, keratogenous and keratinisation portions. Gel-free proteomic analysis of dissected portions from 30 follicles showed that the first keratins to appear were K31, K35 and K85, in the bulb portion. The first epithelial KAP, trichohyalin, was detected in the bulb portion and the first cortical KAP, KAP11.1 was found in the elongation portion. Other major trichocyte keratins and cortical KAPs began to appear further up the follicle in the keratogenous and keratinisation zones. These results were consistent with what has been observed from gene expression studies and correlated well with the morphological changes observed in the follicle. Other proteins detected by this approach included the keratin anchor protein desmoplakin, as well as vimentin and epithelial keratins, histones, ribosomal proteins and collagens. Two-dimensional electrophoretic (2DE) analysis of dissected portions of 50 follicles revealed substantial changes in the position, number and intensity of the spots of the trichocyte keratins as they progressed through the follicle zones, suggesting that they are subject to modification as a result of the keratinisation process. Also present in the 2DE maps were a number of epithelial keratins, presumably from the inner and outer root sheaths, and the dermal components.