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

Journal of Structural Biology 166 (2009) 46–58

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Cortical cell types and intermediate filament arrangements correlate with fiber curvature in Japanese human hair Warren G. Bryson a,*, Duane P. Harland b, Jonathan P. Caldwell c, James A. Vernon b, Richard J. Walls b, Joy L. Woods b, Shinobu Nagase d, Takashi Itou d, Kenzo Koike d,* a

Formerly of Canesis Network Limited, Now a Consultant at 55 Westlake Drive, Halswell, Christchurch 8025, New Zealand Formerly of Canesis Network Limited, Now at AgResearch Limited, Lincoln Research Center, Private Bag 4749, Christchurch 8025, New Zealand c Formerly of Canesis Network Limited, Now at Environment Waikato, P.O. Box 4010, Hamilton, New Zealand d Beauty Research Center, Kao Corporation, 2-1-3, Bunka, Sumida, Tokyo 131-8501, Japan b

a r t i c l e

i n f o

Article history: Received 11 September 2008 Received in revised form 11 December 2008 Accepted 11 December 2008 Available online 25 December 2008 Keywords: Human hair curvature Cortical cell classification Morphology Ultrastructure Electron microscopy and tomography Fluorescence light microscopy Intermediate filament arrangements

a b s t r a c t 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. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction The appearance of human scalp hair contributes to the perception of an individual’s ethnicity, religion, culture, and individuality. A key determinant of hair style is the amount of waviness or curl (Robbins, 1994) contributed from the curvature of individual fibers. The human hair fiber can be described as a long, thin, cylindrical, and flexible shaft consisting of a core covered by relatively thin and flat, but circumferentially curved, overlapping cuticle cells. The core, or cortex, is composed of elongated, keratinized cells aligned, or slightly inclined with the direction of the longitudinal fiber axis, and often contains a centrally located, strand of highly vacuolated hardened cell remnants known as medulla cells (Orwin, 1979a). Remarkably, there is insufficient human hair structural biology information known to provide a fundamental understanding of hair curvature. In contrast, substantial progress in sheep wool fiber structural biology (Rogers, 1959a,b; Bradbury, 1973; Kaplin and Whiteley, 1978; Orwin, 1979a,b; Marshall et al., 1991; Bryson * Corresponding authors. Fax: +81 3 5630 9326 (K. Koike). E-mail addresses: [email protected] (W.G. Bryson), [email protected] (K. Koike). 1047-8477/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2008.12.006

et al., 2001; Caldwell et al., 2005a,b) reveals a strong correlation between the lateral distributions and abundances of para-, meso-, and orthocortical cell types and single fiber curvature (Horio and Kondo, 1953; Fraser and Rogers, 1955; Whiteley and Kaplin, 1977; Kaplin and Whiteley, 1978) and fiber diameter (Orwin and Woods, 1980; Orwin et al., 1984). Transmission electron microscopy (TEM) has revealed that cortical cell types are differentiated by morphological and/or structural variations in distinct subcellular components, namely: the intermacrofibrillar material (IMM), the cytoplasmic remnants (CR), the macrofibrils (Mfs), and the Mf components: the intermediate filaments (IFs) or trichocyte (hard a-keratin) proteins, embedded in a matrix material composed of keratin associated proteins (KAPs). Mfs occur as elongated bodies (length maximum approximately () 10 lm) aligned, or slightly inclined, within the cortical cells, in the direction of the fiber’s longitudinal axis. IFs are 7 to 10 nm in diameter, of length 1 lm, and are aligned longitudinally within Mfs (Bradbury, 1973). IFs are comprised in cross-section of assemblies of 32 individual, longitudinally aligned, IF proteins which form a molecular sub-unit 47 nm long (Steinert, 1993; Wang et al., 2000). Neighboring molecular sub-units link near the juxtaposed head and tail regions of the individual IF proteins to form a single supra-molec-

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ular chain which comprises the IF. In contrast, the matrix surrounding individual IFs is an amorphous cystine-rich proteinaceous material. Its protein composition (Plowman et al., 2007) and chemical properties vary with cortical cell types, in its reaction with reductive and oxidative reagents, and in the absorption of staining reagents for light and electron microscopy (Bradbury, 1973; Zahn, 1980; Jones et al., 1990). Matrix staining variability and IF arrangements are useful identifiers of cortical cell types (Horio and Kondo, 1953; Rogers, 1959a; Bonés and Sikorski, 1967; Kaplin and Whiteley, 1978; Orwin and Bailey, 1988). Fiber curvature has been hypothesized to result from the interaction between different 3D IF arrangements (inferred from 2D TEM images) and the matrix material following dehydration as the fiber emerges from the follicle (Munro and Carnaby, 1999; Munro, 2001). Electron tomography directly confirmed the different IF arrangements in wool cortical cell types (Bryson et al., 2000; Caldwell et al., 2005a,b), enabling further mathematical modeling and computer assimilations to test hypotheses predicting the effect of IF arrangements on single fiber curvature (Bryson et al., 2001; Liu and Bryson, 2002, 2005). Few studies have aimed to investigate specifically the structural basis of human hair curl. Two comparative studies, from the same laboratory, of six human hair samples, varying from straight (Caucasian) to highly curled (African), indicated that some structural and compositional features relate to hair curl (Barbarat et al., 2005; Thibaut et al., 2007). Fiber ellipticity values were positively, but not always consistently, correlated with an increase in curliness. Both studies used TEM to correlate differences in the abundances of ortho-, meso-, and paracortical cell types, as previously defined for wool, to the degree of hair curl. The identification of cell types was not confirmed with high-magnification data. Small-angle X-ray scattering (SAXS) measurements of the axial stagger between IFs indicated that the average 67 Å arc position increased when progressing from straight to highly curved hairs, and that its dispersion increased with the degree of curliness (Barbarat et al., 2005) (implying increased IF tilt). A related study (Kajiura et al., 2006) used SAXS to investigate the internal nanostructure in five hair types varying from straight to highly curved fibers. The equatorial and azimuthal scattering intensity profiles indicated IF orientation and packing differences between the cortical regions adjacent to the inside and outside surfaces of the hair curve. They concluded that while curved hairs have a bilateral distribution of ortho-like and para-like cortical cell types, straight hairs have a homogeneous mixture of these cell types. Japanese women’s hair, although commonly regarded as being straight, varies in curl radius from 0.6 to 16.0 cm with a mean of 4.4 ± 2.3 cm (Nagase et al., 2008). In curved fibers, an imaginary curved plane orientated perpendicularly to the direction of curvature, divides the fiber, centrally through the medulla, into two halves—one located adjacent to the concave fiber surface and the other adjacent to the convex surface (Fig. 1). TEM revealed the presence of different cellular morphologies within these laterally opposed cortical halves (hereafter termed the concave and the convex halves). The differing Mf arrangements, internal structures, and 2D IF arrangements, and their relative locations in the dominant cell types, appeared similar to those of the ortho- and paracortical cells of wool. However, their distinct 3D IF arrangements could not be confirmed. In this study, microscopy techniques were used to define the structural biology features that characterized naturally straight and curved Japanese women’s hair. A classification scheme is described for human hair cortical cells defined by their TEM appearance. This scheme identified some cell types different to those found in wool. The abundances and distributions of the different cell types in relation to fiber curvature were determined using TEM and a newly developed fluorescence light microscopy (FLM)

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method. Electron tomography enabled 3D reconstruction of IF arrangements in the predominant cell types. A possible mechanism for how cell distributions, IF arrangements and the matrix material influence hair curvature is described.

2. Materials and methods 2.1. Human scalp hair samples Sets of naturally straight and curved hairs were collected from adult Japanese women donors (Nagase et al., 2008) and supplied to Canesis Network Limited (now AgResearch Limited), New Zealand, by Kao Corporation, Japan. Donors claimed that their hair had not encountered harsh chemical or physical treatments within the past six months. 2.2. Determination of the relative proportions of cellular components in hair fibers Transverse sections (1.5–2 lm thick) of unstained hairs were examined with optical microscopy (Zeiss microscope, (Carl Zeiss, Germany), DP70 digital camera (Olympus), and DP Controller software), and digital images were captured. SigmaScan software (Systat Software Inc., USA) was used to automatically detect and calculate melanin granule areas, based on shape and size parameters and stain density threshold values. Cuticular and medulla areas were calculated from manual tracings. Conversion of areas to volumes assumed that cellular components were thicker than fiber sections. 2.3. Measurement of single fiber curvature in human hair Hairs of length >140 mm were relaxed in water (10 min, 20 °C) to remove temporary set, and air-dried on vibrating filter paper. Lengths of hair required for TEM were cut 30 mm from the fiber root-end, constrained in 2D with a transparent plastic sheet and digitally photographed. The fiber images were reduced to a one pixel line-width, and curl diameter was measured by fitting three equidistant points along the 5 mm segment using AnalySISÒ Pro V 3 software, (Soft Imaging Systems, GmhB, Germany). 2.4. Classification of human hair cortical cell types by TEM The root-ends of three straight (curl diameter >120 mm) and two curly (curl diameters of 51.4 and 17.5 mm) hair fibers were secured on acetate plastic frames (Nelson and Woods, 1996), then chemically reduced and stained with osmium tetroxide and uranyl acetate (Orwin et al., 1984). The dehydrated fibers were embedded in LR White acrylic resin (London Resin Co., Reading, U.K.) and ultrathin (60–90 nm) transverse sections were cut (Reichert-Jung Ultracut E ultramicrotome) and imaged using a TEM (EM 300, Philips, Netherlands). Negatives were digitally scanned (2400 dots per inch) and fiber cortical cell types were classified on the basis of their morphology and ultrastructure. 2.5. Distribution of cortical cell types in straight and curved human hairs The distributions and proportions of the different cell types in transverse fiber sections were correlated with the direction and the degree of fiber curvature by the following procedures. 2.5.1. TEM mapping of cell types Segments, 5 mm long, 30 mm from the root-end of three straight and three curved fibers were embedded in acrylic resin,

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Fig. 1. Cortical cell structure in a curved human scalp hair. A plane of curvature (P) aligned perpendicular to the direction of curvature bisects the hair fiber into two halves: concave and convex. Electron microscopy reveals a core structure with a surrounding layer of stacked, overlapping, tile-like cuticle cells and a centrally located medulla (M). The cortex contains different cell types separated by a cell membrane complex (arrowheads). Cortical cells are composed mostly of bundles of keratin intermediate filaments called macrofibrils () which may be discrete or fused. Cortical cells often have a central cytoplasmic remnant (CR) and melanin granules (MG) are apparent as circular black structures dispersed throughout the cortex.

and ultrathin transverse sections were cut for TEM. The sections were sequentially stained with 2% uranyl acetate and 0.2% lead citrate, then examined with a TEM (Morgagni 268D, FEI, USA) at 80 kV and 18,000 times () magnification. Photographic montages were collated from micrographs taken with a SIS Megaview III digital camera covering a sector 25 lm wide across the minor lateral axis of each fiber section. Cortical cells were classified and colorcoded in each montage to produce a distribution map (Coreldraw V11), which was rescaled for comparison with a FLM image of an adjacent thick section of the same fiber. 2.5.2. Fluorescence microscopy of cortical cell types Transverse sections (1.5 lm thick) of the above hairs were cut within 14 lm of the ultrathin sections and dried onto PolysineTM coated slides. A dual fluorescence staining procedure, developed from a wool cortex cell sorting method (Ley et al., 1990), was applied as follows. In a dark-room, the slides were immersed in a phosphate-buffered 0.002% fluorescein sodium (FS) solution for 18 h, water rinsed, dried, then immersed in a phosphate-buffered 0.0005% sulforhodamine 101 (SR) solution, water rinsed, dried, and cover-slipped with an anti-fade mountant (DAKO Ltd.). Sections were immediately observed by FLM with a Zeiss Standard 14 light microscope fitted with a Zeiss Plan-Neofluar 40 objective lens (1 mm aperture), a mercury lamp (80 W) and a fluorescein filter set (BP 450–490, FT 510, LP 520). Digital micrographs were taken within 10 s of initial exposure to the mercury lamp. Cortical cell types corresponding to the green (FS stained) and red (SR stained) fluorescent regions were identified from TEM images of adjacent ultrathin sections. 2.5.3. Sheep wool fiber samples Unprocessed, high-curvature (curl diameters ranging from 1.3 to 2.2 mm) wool fibers sampled from the mid-side of five individual New Zealand Merino sheep were prepared in a similar manner to the human hair samples for the FLM and TEM imaging of trans-

verse fiber sections to establish the specificity of the two fluorescent stains, FS and SR, for the ortho- and paracortical cells found in wool. 2.6. Electron tomography for determination of 3D IF arrangements in cortical cell types Fiber segments (5 mm long) from one straight and two curved hairs were stained by immersion in 5% aqueous silver nitrate solution (Tester, 1987) for 6 h. Excess silver was removed by water rinsing for 30 s. Segments were embedded in Spurr’s low viscosity epoxy resin (EMS, USA). Transverse sections, 150 nm thick, were cut (Ultracut UCT ultramicrotome, Leica, Germany) with a 35° diamond knife (Diatome, Switzerland). Fiber sections were labeled with fiducials (15 nm mean diameter) by immersion in a colloidal gold solution (Ted Pella Inc., USA). An intermediate voltage TEM (Tecnai G2 30, FEI, USA), operating at 300 kV was used for imaging Mfs in the different cortical cell types. A tomogram of each Mf, and its immediate surroundings, was constructed from the assembly of a set of 87 tilt-series images collected from 64.5° to +64.5° by sequentially tilting the fiber section in 1.5° increments around the specimen axis perpendicular to the electron beam. Images (2 lm2, pixel size 0.93 nm) were collected with a Tietz digital camera (2048  2048 pixels, 12 bit) at a microscope magnification of 13,500. Each set of tilt-series images was processed using the IMOD software program suite (http://www.bio3d.colorado.edu; Kremer et al., 1996) to construct a tomogram of the 3D structural data within the selected fiber volume, as previously described for wool fibers (Bryson et al., 2000; Caldwell et al., 2005a,b). Gold fiducials spread over both sides of the region of interest enabled IMOD to accurately align the tilt images, and to correct for image distortions and section thinning in the electron beam. 3D IF arrangements within each Mf were modeled by tracing individual IFs on either side of an IF located at the center of a

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region of hexagonally packed IFs, to form a row of IFs extending towards the opposed peripheral Mf boundaries. Once modeled, IFs could be displayed to observe their pitch angle and orientation from any direction. The IMOD program, Fiberpitch, was used to determine IF coordinate data. The cosine of the IF helical tilt angle, a, was calculated as the length, x, of the IF portion traced within the tomogram divided by the tomogram thickness, y. Plotting graphs of a versus IF distance, d, from the central IF, and determination of their slopes, provided a comparative measure of the degree of IF helicity in Mfs within and between the cell types in hair fibers of different curvatures. The mean IF slope data were compared using unpaired two-tailed t tests with 95% confidence intervals.

3. Results 3.1. Condition of hair samples The curved hairs examined with TEM showed signs of minor damage, including the loss of some outermost cuticle cells, small holes in the endocuticle and CR, gaps around most melanin granules, and some splitting along the cell membrane complex. Such minor damage did not structurally alter the cellular features used for cortex cell classification, nor could it change IF arrangements. However, chemical damage has potential to affect the staining characteristics of TEM and fluorescent stains, and precautions were taken to exclude hair samples where such damage was apparent. In straight hairs examined with TEM, holes were uncommon, and cell membrane splitting was almost non-existent. 3.2. Classification of cell types in straight and curved human hair Fig. 1 provides an overview of the human hair fiber at several structural levels and defines some of the geometrical features and terms used to describe fiber curvature and morphology. TEM of transverse fiber sections revealed variations in the morphology and ultrastructure of the cortical cell components (Fig. 2). At low magnifications (4000–9000), Mfs appeared as distorted-circular structures generally 200–500 nm in diameter. The packing of Mfs within cells varied from appearing to be fully separated by IMM to almost completely fused, to form larger irregular-shaped regions. Primary criteria for morphological differentiation of cell types were the extent of Mf separation, the relative size and shape of Mfs, and the characteristics (networked, large, medium, small, present or absent) of the CR. At high magnifications (80,000– 120,000) each Mf was seen to contain hundreds of IFs, aligned approximately parallel with the longitudinal fiber axis, in apparent end-on or tilted arrangements (Fig. 2B and D). The ultrastructure of the Mfs was also an important criterion for cell classification. It was characterized by hexagonally close-packed or pseudo-hexagonally loose-packed IFs in apparent spatial arrangements varying from either extended parallel arrays to distorted helices which often became whorl-like near the peripheral regions of the Mf. Criteria observed at both low and high magnifications enabled the identification of four discernable cortical cell types, provisionally named as Types A, B, C, and D (Fig. 2), all found in straight and curved hairs. Type A cells typically contained Mfs that were well separated by a distinct and continuous layer of IMM, but a distinct CR was absent (Fig. 2E). The Mfs tended to be approximately circular or rounded structures, mostly similar in size and relatively loosepacked in lateral view. Near the center of Mfs, IFs appeared to be hexagonally packed and oriented approximately parallel to the fiber axis. Away from the center, IFs appeared to be tilted in helical arrangements similar to those found in the Mfs of the Type B cells (Fig. 2A and B) and the orthocortical cells of wool.

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In Type B cells (Fig. 2A), Mfs tended to be more close-packed and to have more variation in size and shape than in Type A cells. Type B Mfs appeared as distinct, approximately circular or elliptical structures, often distorted in shape by packing against neighboring Mfs, but clearly separated by a thin border of IMM. CR appeared in 15% of Type B cells. Type B Mfs (Fig. 2B) typically had a small region of hexagonally packed IFs appearing end-on near the central region of each Mf, which appeared to progressively tilt into helical IF arrangements then, into concentric whorl-like bands near the Mf perimeter (this IF arrangement is referred to as helical/whorl-like, hereafter). Some Mfs contained two or three helical/whorl-like structures. Many of the hexagonally packed IF patterns appeared displaced off-center. This may result from transverse sectioning if the embedded fiber was not exactly perpendicular to the cutting direction or it may indicate that these Mfs lie inclined to the fiber longitudinal axis. In such cases the whorl-like appearance is exacerbated. Type C cells were characterized by relatively large Mfs (Fig. 2C). A centrally located CR was found in 40% of the Type C cells. Neighboring Mfs were either in direct contact (without a thin separating layer of IMM), or they were fused together such that their borders were poorly discernable or indistinguishable. IMM was generally present at junctions between three or four Mfs. Apparent shading differences distinguishing the boundaries of fused Mfs were attributable to counter directional IF arrangements, the latter observable at high magnifications. Large Mfs often exhibited hexagonally packed IFs in helical arrangements with whorl-like outer regions (Fig. 2D lower panel) along-side extended close- or loosepacked hexagonal IF arrays (meso- and para-like) aligned approximately parallel with the fiber longitudinal axis (Fig. 2D, upper panel). Type D cells were the least common cell type. They had relatively large Mfs that were well separated by, and contained up to 50% of, CR material and/or IMM (Fig. 2F). They were always found adjacent to Type C cells, and had a similar mixture of IF arrangements. 3.3. Lateral distributions of cortical cell types in straight and curved hairs TEM mapping of cell types in transverse hair sections revealed that their distributions differed between straight and curly hairs. In straight hairs (Fig. 3A), cell types were distributed in approximately symmetrical annular bands around the medulla. Cells adjacent to the cuticle were almost exclusively Type A or B cells. Further toward the medulla, the cortex contained a mixture of primarily Type B and C cell types and a few Type D cells. Type B cells, and to a lesser extent, Type A cells, were predominant directly adjacent to the medulla. In curved hairs (Fig. 3B), TEM mapping revealed a strong bilateral asymmetry in the distribution of Type B and C cells in the cortex on either side of the medulla, with a plane of symmetry approximately aligned in the direction of hair curvature. The cortex between the convex cuticle surface and the medulla was composed almost entirely of Type B cells with few inter-dispersed Type C or D cells. In contrast, the cortex between the medulla and the concave cuticle surface (Fig. 3B) was dominated by Type C cells with few inter-dispersed Type B cells. Type D cells were rare. Typically, a thin band of Type A and B cells was located adjacent to both the convex and concave fiber surfaces. Type B cells and the occasional Type C cell were found immediately adjacent to and surrounding the medulla. The FLM method also revealed differences in the cortices of straight compared with curved hairs (Fig. 4A). Comparison of FLM sections with adjacent sections processed for TEM indicated that staining differences occurred at the whole-cell level, with some individual cells being stained green and other cells red

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Fig. 2. Subcellular structure and ultrastructure of human hair cortical cell types. Transmission electron micrographs of sections were stained by reduction/osmication followed by uranyl acetate and lead citrate. (A) In Type B cells, Mfs appear as distorted-circular structures, discretely separated by IMM (arrowheads). A melanin granule (MG) appears as a black circular structure. (B) Individual Mfs of Type B cells, at high magnification, typically reveal a central region of lightly stained IFs, as hexagonally packed dots in an electron dense matrix. The IF packing changes to an apparent helical conformation then to a whorl-like pattern towards the periphery of the Mf. (C) In Type C cells, Mfs are larger, fused at their boundaries and arranged around an electron dense cytoplasmic remnant (CR). (D) Type C cell Mfs exhibited pseudo-hexagonal IF packing, varying from loose- (upper panel) to close-packed (Fig. 8A-a) IFs in arrays aligned approximately parallel with the Mf axis, to helical/whorl-like (lower panel) arrangements as observed in Type B cells. (E) Type A cell Mfs are typically similar in size, more circular than Mfs in Type B cells, well separated by a layer of IMM (arrowheads) and have Mfs containing IFs in helical/whorl-like arrangements. (F) In Type D cells, up to 50% of the transverse area is CR or IMM material. The fused Mfs contain IF arrangements similar to those found in Type C cells. Scale bars: 0.5 lm for A, C, E, and F; 100 nm for B and D.

(Fig. 4B). Stain color was found to correlate to the cell type, as determined from TEM, across the cortex along the direction of curvature (typically across the minor lateral fiber axis) (Fig. 5). FS preferentially stained Type C cells and SR preferentially stained Type A and B cells. Fluorescent image data were similar to those more laboriously produced from TEM micrographs. In straight hairs,

SR-stained and FS-stained cells were approximately symmetrically and annularly distributed throughout the cortex. The SR-stained cells were in highest abundance close to the medulla and close to the cuticle cells (Figs. 4A and 5). Curved fibers always revealed a bilateral distribution with a greater abundance of FS-stained (Type C) cells located in the concave fiber half, and a greater

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Fig. 3. Cortical cell distributions mapped from TEM micrographs of straight and curved hair. The lateral distributions of the four cortical cell types (right) were mapped from high-magnification micrographs of the fiber sections (left). Sections were stained with reduction/osmication followed by uranyl acetate and lead citrate. (A) Straight hair. A heterogeneous distribution of mainly Type B and C cells dominated much of the cortex with a higher predominance of Type A and B cells close to the cuticle and the medulla. These cell types formed annular lateral distribution patterns in the overall cortex. (B) High-curvature hair. An asymmetric bilateral distribution of cell types is evident relative to the direction of fiber curvature (arrow). Type C cells predominated in the concave fiber side, whereas, Type B cells were predominant in the convex fiber side.

abundance of SR-stained (Type A and B) cells located in the convex fiber half (Figs. 4B and 5). These FLM results were reproducible for all fibers examined. Comparison of FLM and TEM images (Fig. 4E), obtained from adjacent pairs of transverse sections cut from high-curvature, Merino wool fibers, revealed in each case that the green FS-stained cortical cells corresponded to the position of the paracortical cells located in the fiber half closest to the concave fiber surface whereas the red SR-stained cells corresponded to the position of the orthocortical cells located in the fiber half closest to the convex fiber surface. Whilst the cell types of wool were more intensely

stained than those of human hair, the staining patterns suggest that the Type C cells of human hair have chemical affinity characteristics similar to the paracortical cells of wool, and the Type A and B cells have chemical affinity similar to the orthocortical cells. 3.4. 3D IF arrangements in Type B and C cells of straight and curved hairs 3.4.1. Modeling of Mfs with helically arranged IFs Mfs with apparently helical IF arrangements were modeled to enable comparisons between such arrangements in Type B and C

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Fig. 4. Fluorescently stained sections of straight and curved scalp hair and curved wool fibers. (A) Transverse hair sections stained with SR and FS revealed the distributions of Type A and B (red) and Type C (green) cells, respectively. In straight hair (left), the stain pattern is relatively uniform across the cortex whereas in the high-curvature hair (right) green staining dominates the concave fiber half and red staining dominates the convex half. (B) A fluorescently stained section with some features matched to uranyl acetate/ lead citrate stained TEM micrographs (C and D) obtained from an adjacent fiber section. (C) A distinctive cell with a high melanin content served as a point of reference between TEM and fluorescent sections. (D) A distinctively shaped bright green cell in the fluorescent section (B) is matched to a Type C cell surrounded by Type B cells in this TEM micrograph. (E) A representative transverse section from a high-curvature wool fiber (left) revealed the distributions of cortical cells stained with FS (green) and SR (red). Comparison with a uranyl acetate/lead citrate stained and TEM imaged, adjacent section of the same fiber (right) revealed that the FS-stained cells correspond to the paracortex and the SR-stained cells to the orthocortex of wool. Asterixis () in the TEM wool image indicate folds in the ultrathin section. Arrows indicate the direction of curvature.

cells, as follows: six 3D IF arrangements in six Mfs were modeled from six reconstructed tomograms obtained from a single, naturally straight hair, and twelve 3D IF arrangements in 12 Mfs were modeled from ten reconstructed tomograms from two curved hairs. Mfs were selected for reconstruction if they were averagesized (200–300 nm diameter), symmetrically rounded, and had apparent helical IF arrangements which included a single, small region of hexagonally packed IFs near the Mf center. Mfs distorted by a neighboring melanin granule or CR, and multi-centered Mfs were avoided.

Representative tomograms and models of Mfs with helical/ whorl-like IF arrangements in silver stained Type B and C cortical cells from a straight hair are presented in Fig. 6A, and from the two curved hairs in Fig. 6B. The models from both the discretely separated Type B cell Mfs and the fused Mfs of Type C cells revealed that the IFs tilt tangentially, and not radially, with tilt angle progressively increasing from the center towards the Mf perimeter. Views from above single rows of IFs modeled laterally across the Mf diameter (images b and f in Fig. 6A and B) and side-on views perpendicular to the row of modeled IFs

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Fig. 5. Cortical cell distributions in adjacent TEM and FLM hair sections. Comparison of cell type maps from TEM micrographs with fluorescently stained neighboring sections from the same fiber revealed near one-to-one cell type matching and identical cell type distribution patterns. (A) Straight hair. Both TEM and FLM reveal a similar, annular Type B and C cell distribution pattern around the cortex. (B) Curved hair. The asymmetric cell distribution pattern reveals that Type B cells predominate in the convex fiber side and Type C cells predominate in the concave side. Arrowheads indicate curvature direction.

(images c and g), confirm the absence of radial tilt, whereas images b and f and the side-on views in-line with the same row of IFs (images d and h), confirm the presence of the tangential tilt of the individual IFs. Helically arranged IFs were observed to have approximately hexagonally packed centers. When the fiber section was tilted so that IFs near the perimeter of the helically packed region could be observed end-on, the pseudo-hexagonal packing was seen to be retained. The Mf models confirm, at least over the width modeled (up to 200 nm) and the depth of a tomogram (150 nm), that an individual IF follows a helical pathway, tilted tangentially as if on an imaginary cylindrical surface scribed by an approximately constant helical tilt angle, a, at a constant distance, d, from the centrally se-

lected longitudinal Mf axis. Being helical, Mfs have chirality (or handedness). Of the 18 Mfs examined, seventeen were left-handed, and one, from a Type C cell in a curved hair was right-handed. Graphs of coordinate data from modeled IFs (Fig. 7) show that the IF helical tilt angle, a, increases approximately linearly with distance, d, from the Mf center, as described by the equation of each graph. The value of a can be calculated at any value of d, such that, in Fig. 7A-c, at a distance of 60 nm from the Mf center, a is 17.0°. The slope of this graph is 0.30 degrees per nanometer (°/ nm), representing the change in a from the center of the Mf to the most peripherally modeled IFs. In the following section, mean IF slope values are used to compare the helical character or ‘helicity’ of Mfs between different cell types.

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Fig. 6. Electron tomograms and models of helically arranged IFs. Representative tomograms and models of helically arranged IFs in Mf regions from Type B and C cells from straight, Fig. 6A, and curved, Fig. 6B, hair. Images a and e show 2D views of tomograms from Type B and C cells, respectively, with the IFs selected for modeling indicated in red. Models of IFs from Type B cells are shown in images b–d, and from Type C cells in images f–h. Views b and f look down on a single row of IFs; c and g are side-on views perpendicular to the row of IFs; and d and h are side-on views along the row of IFs.

IF slope values derived from individual Mfs with helically arranged IFs were highly variable, ranging from 0.19 to 0.42°/nm in Type B cells and from 0.16 to 0.56°/nm in Type C cells (Fig. 7). There was no significant difference between the overall mean IF slope obtained from six helical Mf regions in a straight hair (0.29 ± 0.07°/nm) and that from the 12 helical Mfs regions in two curved hairs (0.32 ± 0.11°/nm). In the straight hair, the mean IF slope in three Type B cell Mfs (0.25 ± 0.06°/nm) was less than that of the three Type C cell Mfs (0.33 ± 0.06°/nm). For the two curved fibers, the mean IF slope in six Type B cell Mfs (0.34 ± 0.06°/nm) was not significantly different from that of the six Type C cell Mfs (0.30 ± 0.15°/nm). In Type B cells, the mean IF slope of three Mfs (0.25 ± 0.06°/nm) from the straight hair, was significantly lower (P < 0.05) than that of the six Mfs from the two curved fibers (0.34 ± 0.06°/nm), whereas the mean IF slope for Type C cell Mfs did not differ between the straight hair (0.33 ± 0.06°/nm) and the two curved hairs (0.30 ± 0.15°/nm). 3.4.2. Modeling of Mfs with IF arrangements in non-helical pseudohexagonal parallel arrays Whereas 2D TEM revealed that all IFs in Type B cell Mfs were variously arranged, helically with whorl-like outer regions, in Type C cell Mfs, IFs varied from such helical/whorl-like arrangements to pseudo-hexagonal or hexagonal parallel arrays, with all IFs aligned with the longitudinal fiber axis. Mfs with apparently non-helical 3D IF orientations were modeled in Type C cells from a curved hair,

for comparison with Type C cell Mfs with helical IF arrangements. Mf regions selected for reconstruction contained fused Mfs with apparently extended regions of loosely or tightly, hexagonally packed IFs. Direct evidence is presented in Fig. 8 for the presence of non-helical IF arrangements in Type C cell Mf regions from a curved hair. The lateral view of the first tomogram (Fig. 8A-a) revealed that IFs were pseudo-hexagonally packed. Tangential and radial IF tilt were absent in the overhead view (Fig. 8A-b) and in the in-line side view (Fig. 8A-d) of the row of IFs. In the side view, perpendicular to the row of IFs (Fig. 8A-c), the IFs were aligned almost parallel to one another. These models confirmed the presence of pseudo-hexagonally packed IFs arranged in a longitudinally aligned, extended parallel array. The second tomogram (Fig. 8A-e) of a Type C cell Mf suggests an apparently very weak helical arrangement of the pseudo-hexagonally packed IFs. When modeled, the apparent IF arrangement is confirmed. An overhead view of the row of IFs (Fig. 8A-f) reveals very slight tangential tilt, but an absence of radial tilt, and in the in-line side view (Fig. 8A-h), the weak tangential tilt is more obvious. In the perpendicular side view (Fig. 8A-g) IFs are predominantly aligned almost parallel to each other. The IF slope values for the two Mf tomograms (Fig. 8A-a and e) of 0.08 and 0.15°/nm, respectively, calculated from the graphs (Fig. 8B-a and b), were substantially less than the mean values of 0.31°/nm determined from Type C cell Mfs with helically arranged IFs (Fig. 7).

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Fig. 7. Representative graphs of IF helical angle versus IF distance from the Mf center. Graphs were plotted using IF helical tilt angle, a, and distance, d, coordinate data obtained from IF models in Mf regions from Type B (graphs a–c) and C (graphs d–f) cells from a naturally straight fiber, Fig. 7A, and two curved fibers, Fig. 7B.

3.4.3. Relative proportions of non-Mf cellular components in hair fibers Optical imaging of hair revealed that the fiber volume contained, on average, 14.3% cuticle, 3.1% medulla, and 8.6% melanin granules (Fig. 1). The melanin granules were rare in the cuticle being mainly confined to the cortex. The cortex of hair, including both the medulla and melanin granules, by calculation, comprised 85.7% of the fiber volume. The imaging method did not yield accurate data for calculation of CMC, CR, and IMM contents. 4. Discussion and conclusion 4.1. Cortical cell structure and classification TEM observation of morphological and structural features in scalp hairs from Japanese women enabled the classification of the cortical cells into four distinct types, A, B, C, and D, found in both naturally straight and curved fibers. Type A and B cortical

cells exhibited Mf and IF arrangements similar to those found in the orthocortex of wool fibers. The Type C and D cells contained fused Mf arrangements similar to the meso- and paracortical cells of wool but differed in that the latter did not contain helical/whorllike IF arrangements (Kaplin and Whiteley, 1978; Caldwell et al., 2005a,b). Type B and C cells were predominant in the cortex, and differences in their lateral distributions were apparent in straight and curved hairs. TEM and FLM revealed an annular cortical cell type distribution pattern in straight fibers, implying that averaged structural differences of Type B and C cells would maintain a straight fiber conformation. In contrast, all curved fibers contained a bilateral distribution of Type B and C cells, with Type C cells predominating in the cortical half adjacent to the concave fiber side. This asymmetric distribution of the predominant cell types, coupled with their differences in subcellular morphology and ultrastructure, is considered to provide a mechanism for fiber

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Fig. 8. Non-helical and weakly helical IF arrangements in Type C cell Mfs from a curved fiber. (A) Tomograms and IF models were constructed from apparently non-helical Mf regions. Images a and e show overhead views of tomograms, with the IFs selected for modeling indicated in red; images b and f are views from above a single row of modeled IFs; c and g are side-on views perpendicular to the row of modeled IFs; d and h are side-on 2D views along the row of modeled IFs. (B) Graphs a and b show IF helical tilt angle versus IF distance from the Mf center plotted using coordinate data obtained from IF models in A, b–d and f–h, respectively).

curvature. These observations support the conclusions reached from SAXS data (Kajiura et al., 2006) and agree with other recent studies of straight and curved hairs (Barbarat et al., 2005; Thibaut et al., 2007; Nagase et al., 2008). Whereas TEM distinguished cell types on the basis of their Mf appearance and IF arrangements, FLM relied on the response of their matrix proteins to the fluorescent dyestuffs; SR and FS. As SR is known to be slightly hydrophobic and FS more hydrophilic, their ability to differentiate Type B and C cells implies the abundance, more or less, of specific KAPs. The orthocortical cells of wool have a matrix rich in high tyrosine/glycine proteins (Plowman et al., 2007), and react strongly with SR and are structurally analogous to Type B cells, whereas the paracortical cells of wool, have a matrix rich in ultra-high sulfur and high sulfur proteins (Plowman et al., 2007), react strongly with FS and have some structural similarities to the Type C cells, (Fig. 4E). Rogers et al., 2002, have confirmed the expression of high tyrosine/glycine, ultra-high sulfur, and high sulfur KAP mRNA in the central fiber forming compartment of the developing human hair follicle, and they also showed the bilateral mRNA expression of the high tyrosine/glycine KAP8.1 in the cortex of a human beard hair follicle. However, they stated that mRNA expression is no guarantee for concomitant protein expression. Further evidence suggesting bilateral expression of KAPs comes from the amino acid analysis of human curved hair. Cysteine was found to be significantly abundant in the concave fiber half and glycine was slightly richer in the convex fiber half (Nagase et al., 2008) suggesting that certain, as yet undetermined, high tyrosine/glycine, ultra-high sulfur, and high sulfur KAPs exhibit bilateral variance which may relate to differing abundances in Type B and Type C cells. While it remains to be confirmed, via

determination of the primary sequences of proteins extracted from specific cell types isolated from the fully keratinised fiber shaft of human hair, that the Type B cells have a matrix rich in high tyrosine/glycine proteins and that the Type C cells have a matrix rich in ultra-high sulfur and high sulfur proteins, these KAPs possibly have an important role in determining the specific IF spatial arrangements observed in hair cortical cell types. Electron tomography enabled direct 3D IF modeling and confirmed spatial arrangements deduced from 2D TEM images. Modeling provided direct evidence that the Mfs with apparent helical IF arrangements were indeed helical rather than in twisted arrangements by revealing that IF tilt was tangential, and radial tilt was negligible. Comparisons of individual helical/whorl-like Mfs within Type B cells revealed wide variation in their degree of helicity. Earlier work (Caldwell et al., 2005a,b) on wool found relatively lower levels of variation in helicity between the Mfs of the orthocortex. Mean helicity values for Type B and C cell helical/whorl-like Mfs were very similar. However, Type C cell Mfs were predominantly composed of pseudo-hexagonally packed IFs that varied from approximately parallel to slightly helical IF arrays, all longitudinally orientated. Consequently, helicity is considerably lower when averaged across the heterogeneous Mfs of a Type C cell than across the more homogenous (all helical/whorl-like) Mfs of a Type B cell. In this first electron tomography study of human hair, a large number of IFs (N  360) were modeled. However, technical restrictions on the Mf selection method, and on the number of Mfs that could be sampled and modeled, limited the Mfs examined to a statistically small number (N = 18) in relation to the number of variables (two cell types, two fiber types). Despite this shortcoming, the data are valuable because they reveal the high degree of vari-

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ability between the IF slopes in Mfs with helically arranged IFs. Future electron tomography studies of human hair may further refine differences between the structure of straight compared to curved human hair, and hair compared to sheep wool. 4.2. The causative mechanism of curvature formation and behavior Structural considerations imply that although the cuticle cells impose a constraining effect (Parbhu et al., 1999; Bryson et al., 2001; Liu and Bryson, 2002; Caldwell and Bryson, 2005), and the medullary cells may contribute to fiber stiffness, neither is envisaged to contribute a causative mechanism for curvature. In Merino wool, the cell membrane complex contributes 3.3%, and with the IMM and CR, contributes 15.9% to the fiber volume (Bradbury, 1973) without known contributions to curvature. Assuming similar compositions in hair, when taken into account with the cuticle, medulla and CMC contents, the Mfs will comprise 58% of the total fiber or 68% of the cortex volume, and by deduction, are the key composites capable of contributing a plausible mechanism for the explanation of curvature formation and behavior. When correlated with the lateral distributions of cortical cell types in curved human hair, IF arrangements ranged from being predominantly helical in the convex fiber half to a mixture of helical and non-helical in the concave fiber half. Coupled with the inferred differences in matrix protein composition, these are considered to be the only features observed in this study that have potential to cause fibers to be naturally curved. The relationship between cortical cell type distribution, ultrastructure and inferred matrix composition, and the curvature of human hair, is analogous to that discovered in wool fibers. Highcurvature fine wool has a bilateral cortical cell distribution with orthocortical cells containing helically arranged IFs located mainly in the convex fiber half, and paracortical cells containing pseudohexagonally packed, parallel arrays of IFs located in the concave fiber half (Kaplin and Whiteley, 1978; Orwin et al., 1984). Single fiber curvature theory (Dobozy, 1959; Brown and Onions, 1961; Munro and Carnaby, 1999) assumes that as the moisture-saturated wool fiber is extruded from the follicle, the fiber dries, and the matrix protein material located between the water-impervious IFs shrinks laterally. In Mfs with helical IF regions, lateral contraction causes a small decrease in IF helical tilt and a corresponding longitudinal extension of the Mf, but in Mfs with IFs aligned in parallel, such a mechanism is absent and extension is negligible. The resulting differential extension, whereby the orthocortical half extends further than the paracortical half, causes the wool fiber to curve towards the paracortex. The fiber region now known as the paracortex was confirmed to be located adjacent the concave fiber surface over 55 years ago (Horio and Kondo, 1953). In high-curvature human hair, cortical cell types are neither as clearly differentiated, nor as discretely bilaterally separated as in wool. Both the Type B cells of hair and the orthocortical cells of wool, are dominant in their respective convex fiber halves, and are morphologically and structurally similar, with Mfs containing helical/whorl-like IF arrangements. Similarly, Type C and wool paracortical cells are dominant in their respective concave fiber halves, but Type C cells differ by having some IFs in helical/whorllike arrangements as well as in parallel arrays. Consequently, the longitudinal extension differential could be expected to be less between Type B and C cells than that between the ortho- and paracortices of wool. When moisture-saturated hair loses water to equilibrate with ambient conditions, the convex fiber half rich in Type B cells is predicted to extend longitudinally more than the concave fiber half rich in Type C cells, causing the fiber to bend towards the region of highest Type C cell density, giving rise to curvature. This partially explains why the human hair in our study has a much lower single fiber curvature than that of fine wool (curl diam-

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eter typically 1.3–2.2 mm for wool fibers used in this study). In human hair, the larger fibre diameter (80–120 lm compared to 15– 24 lm for fine wool), the higher proportion of medulla, and the greater number of cuticle cell layers (8 compared to 1–3 in wool) all contribute to increased fiber stiffness, which would constrain the bending forces generated by the longitudinal extension differential arising from the bilateral cortical cell type distributions. This study does not preclude other potential contributors to fiber curvature such as faster cell type replication rates, or the formation of longer cells, either of which, if occurring on the convex fiber side, during fiber formation in the follicle, could generate a bilateral length differential resulting in curvature change. The keratinous components of the hair fiber can be thought of as a complex assembly of various proteins polymerized predominantly by the inter- and intra-molecular disulfide bonds of cystine. In practice, as is common in hair perming, it is possible to change the curvature of hair through the chemical cleavage of disulfide bonds via mild reduction, followed by the imposition of stress, and disulfide bond resetting via mild oxidation. A precise knowledge of the protein pairs involved, their molecular conformations and the locations of the coupling cysteinyl pairs, as well as their influence upon the fiber’s ultrastructure, are insufficiently known to enable an understanding of how disulfide crosslinking underpins fiber curvature. Although beyond the scope of this current manuscript, this is a fruitful area of endeavor for future research. In conclusion, in the fully keratinized human hair, differences in the proportions and bilateral distributions of Type B and C cells result in differences in fiber curvature which are considered to be caused primarily by differences between their respective matrix materials and IF arrangements. For a given fiber, current theory predicts changes in curvature can be explained by the swelling or shrinkage behavior of the Mf composite. In the shrinkage scenario, the different IF arrangements in the two prominent cell types is predicted to result in a bilateral extension differential when the matrix material contracts laterally in helically arranged IFs, causing a slight decrease in IF tilt and a slight increase in Mf length, resulting in an increase in hair curvature towards the region of highest Type C cell population. Acknowledgments The authors express their gratitude to the following people for helpful discussions, guidance and support: Masaru Tsuchiyaa, Toshihiko Matsuia, Satoshi Shibuichia, Hisashi Tsujimurab, Yoshinori Masukawab, Naoki Satoha, Koichi Nakamuraa, Naohisa Kurea, Itomi Hommaa, Jeffrey E. Plowmanc and Robert A. McPhersond; and to Kao Corporationa for funding this research. a Beauty Research Center, Kao Corporation, 2-1-3, Bunka, Sumida-ku, Tokyo 131-8501, Japan. b Analytical Science Research Laboratories, Kao Corporation, 2606, Akabane, Ichikai, Haga, Tochigi 321-3497, Japan. c Formerly of Canesis Network Limited, Now at AgResearch Limited, Lincoln Research Center, Private Bag 4749, Christchurch, New Zealand. d Formerly of Canesis Network Limited, Now at FlexiScience, 223 Mulgowie Rd.,Thornton, Qld. 4341, Australia. References Barbarat, P., Fiat, F., Cavusoglu, N., Hadjur, C., Leroy, F., Doucet, J., 2005. From the molecular structure to the macroscopic shape of hair curl patterns. In; Proceedings of the 11th International Wool Textile Research Conference. CD publication 111H, Leeds, UK. Bonés, R.M., Sikorski, J., 1967. The histological structure of wool fibres and their plasticity. J. Text. Inst. 58 (11), 521–532. Bradbury, J.H., 1973. The structure and chemistry of keratin fibres. In: Anfinsen, C.B., Edsall, J.T., Richards, F.M. (Eds.), Advances in Protein Chemistry, vol. 27. Academic Press, New York, pp. 111–211.

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