Known and Unknown Features of Hair Cuticle Structure: a Brief Review

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Commentary Known and Unknown Features of Hair Cuticle Structure: A Brief Review

George E. Rogers

Molecular and Biomedical Sciences, University of Adelaide, Adelaide, SA 5005, Australia; [email protected]

Received: 26 March 2019; Accepted: 9 April 2019; Published: 9 May 2019

Abstract: The cuticle is the outermost layer of overlapping flattened cells of hair and has been subjected to many years of study to understand its structure and how it develops in the follicle. The essential function of the cuticle with its tough inelastic protein content is to protect the inner cortex that provides the elastic properties of hair. Progress in our knowledge of hair came from studies with the electron microscope, initially transmission electron microscopy (TEM) for internal structure and later the scanning electron microscope (SEM) for cuticle surface shape and for investigating changes caused by various environmental influences such as cosmetic treatments and industrial processing of wool. Other physical techniques have been successfully applied in conjunction with proteomics. The outstanding internal features of the cuticle cells are the internal layers consisting of keratin filament proteins and the keratin-associated proteins. The stability and physical toughness of the cuticle cell is partly accounted for by the high content of disulphide crosslinking. The material between the cells that holds them tightly together, the cell membrane complex, consists of a layer of lipid on both sides of a central protein layer. The lipid contains 18-methyleicosanoic acid that is part of the hydrophobic lipid surface of hair. For the past decade there have been aspects that remained unanswered because they are difficult to study. Some of these are discussed in this brief review with suggestions for experimental approaches to shed more light.

Keywords: hair; cuticle; internal structure; protein layers; cell membrane complex; electron microscopy; future research

1. Introduction The cuticle of hair is vital in protecting the inner structure, the cortex, from damage caused by natural environmental factors, cosmetic treatments, industrial processes and the invention of new ones. It is a multiple layer in human hair (single layer in wool) of ﬂattened overlapping and physically hard cells covering the hair cortex (Figure1). The outward orientation of the edges of the cells provides self-cleaning of the continuous desquamation of the epidermis. Since the hair cuticle was studied by early histologists [1] a large literature has developed. It is brieﬂy reviewed here with relevant references to our understanding of cuticle structure achieved through the use of techniques of cell biology. These include transmission electron microscopy (TEM), scanning EM (SEM), X-ray photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS) and atomic force microscopy (AFM) in conjunction with protein analytical techniques. Research over some 70 years has revealed the major details of the internal structure of cuticle cells. It is important to also emphasise that all of these techniques have contributed to the knowledge of hair structure and properties generally.

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(a) (b) Figure(a 1.) (a) SEM of wool fibre ×1500; (b) SEM of hair fibre ×400.(b) FigureFigure 1.1. ((aa)) SEMSEM ofof woolwool fibrefibre ×1500;1500; (b) SEM of hair fibrefibre ×400.400. 2. The Three Layers in a Cuticle Cell × × 2. The Three Layers in a Cuticle Cell 2. TheThe Three outstanding Layers in feature a Cuticle is the Cell three layers, A-layer, exocuticle and endocuticle (Figure 2) that The outstanding feature is the three layers, A-layer, exocuticle and endocuticle (Figure2) that wereThe first outstanding distinguished feature in the is theTEM three by theirlayers, elec A-layetron r,densities exocuticle and and produced endocuticle according (Figure to 2) their that were first distinguished in the TEM by their electron densities and produced according to their relative relativewere first sulphur distinguished contents inbinding the TEM the bymetal their stains elec oftron osmium, densities lead and and produced mercury according for TEM imagingto their sulphur contents binding the metal stains of osmium, lead and mercury for TEM imaging [2,3]. [2,3].relative sulphur contents binding the metal stains of osmium, lead and mercury for TEM imaging [2,3].

Figure 2. TEMTEM of of cross-section cross-section of human hair with five ﬁve overlapping cuticle cells. Each cell has a dense A-layer and a less dense exocuticle beneath. The lower layer is endocuticle. ×35,00035,000. Figure 2. TEM of cross-section of human hair with five overlapping cuticle cells.× Each cell has a dense A-layer and a less dense exocuticle beneath. The lower layer is endocuticle. ×35,000 The layer closest to the upper surface of each cuticlecuticle cell is the densely-stained A-layer then the exocuticleThe layer and beneathclosest to that the theupper endocuticle. surface of each cuticle cell is the densely-stained A-layer then the exocuticleDuring and hair beneath growth that and the protein endocuticle. synthesis, the cuticlecuticle and cortex proteins are synthesised with the sulphur sulphurDuring in inhair the the growth reduced reduced and state state protein as as cysteinyl cysteinyl synthesis, residu residues. thees. cu Asticle a As consequenceand a consequence cortex proteins the metal the are metal stains, synthesised stains, osmic osmic acid,with acid,leadthe sulphur citrate lead citrate inand the and mercuricreduced mercuric stateiodide iodideas cysteinylreadily readily react residu react toes. toincrease As increase a consequence electron electron density the density metal of ofstains, the the keratin keratin osmic acid, and associatedlead citrate keratin and mercuric proteins until iodide cross-linking readily react occurs to increase about half electron way alongdensity the of follicle the keratin whence and the stainingassociated diminishes. keratin proteins A A TEM untilstudy cross-linking of the developing occurs cuticle about cell half [4,5] [4 way,5] showed along thethat follicle the exocuticle whence andthe staining diminishes. A TEM study of the developing cuticle cell [4,5] showed that the exocuticle and

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Cosmetics 2019, 6, x FOR PEER REVIEW 3 of 8 A-layerCosmetics 2019 develop, 6, x FOR as smallPEER REVIEW (Type 1) and large droplets (Type 2) of protein. The small droplets appear3 of to 8 be the precursors of the A-layer and the larger ones, the exocuticle (Figure3). A-layer develop as small (Type 1) and large droplets (Type 2) of protein. The small droplets appear to be the precursors of the A-layer and the larger ones, the exocuticle (Figure 3).

Figure 3. Early stage f formation of A-layer and exocuticle showing type 1 droplets migrated to cell Figure 3. Early stage f formation of A-layer and exocuticle showing type 1 droplets migrated to cell membrane and mixed with filaments (black arrow). The larger Type 2 droplets nearby are in the membrane and mixed with ﬁlamentsfilaments (black arrow). The The larger larger Type Type 2 droplets nearby are in the cytoplasm (arrowheads). From reference [4]. cytoplasm (arrowheads). From reference [[4].4]. These dynamic events imaged in the TEM as static changes suggested that the smaller droplets These dynamic events imaged in the TEM as static changes suggested that the smaller droplets form the outermost A-layer. They migrate and aggregate including with keratin ﬁlaments to form form the outermost A-layer. They migrate and aggregate including with keratin filaments to form the amorphous A-layer beneath the outermost plasma membrane of the cell. The larger droplets as the amorphous A-layer beneath the outermost plasma membrane of the cell. The larger droplets as precursors of the exocuticle increase in size, they also migrate outwards to form a layer of complex precursors of the exocuticle increase in size, they alsoalso migratemigrate outwardsoutwards toto formform aa layerlayer ofof complexcomplex lamellae. In some orientations a sparse central denser layer is discernible in the lamellae (Figure4)[ 6], lamellae. In some orientations a sparse central denser layer is discernible in the lamellae (Figure 4) suggesting that some dense A-layer protein is incorporated into the exocuticle laminae. There is a gap [6], suggesting that some dense A-layer protein is incorporated into the exocuticle laminae. There is in our knowledge here because there are no TEMs showing the development of this event. a gap in our knowledge here because there are no TEMs showing the development of this event.

Figure 4. Late stage of exocuticle (exo) formation in a cuticle cell (FCU) adjacent to inner root sheath Figure 4. Late stage of exocuticle (exo) formation in a cuticle cell (FCU) adjacent to inner root sheath cell (IRSCU) and separated by an intercellular CMC layer (IL). Note the inner dark straining in the cell (IRSCU) and separated by an intercellular CMC layer (IL). Note the inner dark straining in the exocuticle laminathatlaminathat mightmight bebe A-layerA-layer protein.protein. ThisThisstage stage is is markedly markedly di differﬀerentent from from that that in in Figure Figure3. exocuticle laminathat might be A-layer protein. This stage is markedly different from that in Figure Provided3. Provided by Dr. by LDr. Jones. L Jones 3. Provided by Dr. L Jones These structures ﬁnally move to abut the A-layer and fuse with it to form a uniform mass, the exocuticle (Figure5).

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These structures finally move to abut the A-layer and fuse with it to form a uniform mass, the Cosmeticsexocuticle2019 (Figure, 6, 32 5). 4 of 8

Figure 5. A cuticle cell of a wool fibre with almost complete consolidation of the exocuticle (ex) and the denserFigure A-layer5. A cuticle at the cell surface of a wool (arrows). fibre with From almost reference complete [4]. consolidation of the exocuticle (ex) and the denser A-layer at the surface (arrows). From reference [4]. An important discovery in these processes was the presence of fatty acids covalently linked to the cuticleAn important surface. Theydiscovery include in these stearic processes and palmitic was acidsthe presence but especially of fatty an acids unusual covalently branched linked chain to fattythe cuticle acid, anteiso-18-methyleicosanoicsurface. They include stearic acidand (18-MEA)palmitic acids [7–9 ].but This especially finding an explained unusual the branched hydrophobic chain propertiesfatty acid, anteiso-18-methyleicosanoic of the hair surface. According acid tothe (18-MEA) best evidence [7–9]. This 18-MEA finding can explained constitute the up hydrophobic to 70% of the covalentlyproperties boundof the hair fatty surface. acids that According entirely coverto the thebest fibre evidence surface. 18-MEA The abundance can constitute of 18-MEA up to 70% relative of the to thecovalently straight bound chain fattyfatty acidsacids isthat indicative entirely ofcover a particular the fibre function surface. suchThe abundance as stability of the18-MEA hydrophobic relative surfaceto the straight or influencing chain thefatty friction acids on is the indicative surface butof answersa particular to these function questions such need as furtherstability research. of the Thehydrophobic fatty acids surface are attached or influencing by thioester the bondsfriction [9 on] to the a protein surface underlay, but answers yet toto be these certainly questions identified. need Thefurther surface research. lipid ofThe 18-MEA fatty acids was are estimated attached to by be 0.9thioester nm thick bonds [10 ][9] and to wasa protein also imaged underlay, by TEMyet to as be a ‘fuzzy’certainly surface identified. layer The [11]. surface The MEA lipid molecules of 18-MEA are was longer estimated than theto be hydrophobic 0.9 nm thick layer [10] andand couldwas also be foldedimaged to by be TEM accommodated as a ‘fuzzy’ insurface the 0.9 layer nm space[11]. The [12 ].MEA molecules are longer than the hydrophobic layerThe and endocuticle, could be folded compared to be accommodated with the exocuticle, in the is a0.9 completely nm space di[12].fferent layer, a loose amorphous massThe that appearsendocuticle, to be compared cytoplasmic with residues the ofexocuticle protein except, is a forcompletely the presence different of the calcium-bindinglayer, a loose proteinamorphous S100E mass [13]. that Its functionappears isto unknown.be cytoplasmic residues of protein except for the presence of the calcium-binding protein S100E [13]. Its function is unknown. 3. The Epicuticle 3. The Epicuticle The epicuticle was the first sub-structure of the cuticle to be studied in the TEM [14]. Its discovery cameThe about epicuticle when hairswas orthe woolfirst weresub-structure treated withof th chlorinee cuticle saturatedto be studied water in and the wasTEM called [14]. theIts Allwordendiscovery came reaction about [15 when] after hairs the authoror wool who were first treated described with chlorine it. Chlorine saturated was knownwater and to reducewas called the frictionalthe Allworden properties reaction of wool; [15] after its eff theect isauthor to produce who first microscopic described membranous it. Chlorine bubbleswas known on the to surfacereduce butthe doesfrictional not otherwiseproperties destroy of wool; the its fibre effect structure. is to produce The epicuticle microscopic is a derived membranous membranous bubbles structure on the estimatedsurface but to does be 50–70 not otherwise nm thick. destroy Initially the the fibre epicuticle structure. was The thought epicuticle to cover is a derived only the membranous hair surface. Furtherstructure study estimated indicated to be it that50–70 it totallynm thick. surrounds Initially the the surface epicuticle of individual was thought cuticle to cellscover [16 only]. The the origin hair ofsurface. its composition Further study is uncertain. indicated it It that is mainly it totally protein surrounds [17] andthe surface the covalently of individual bound cuticle lipids, cells mainly [16]. 18-MEA,The origin are of cleavedits composition from the is surfaceuncertain. by It the is chlorinemainly protein [9]. The [17] A-layer and the is covalently thought to bound be involved lipids, becausemainly 18-MEA, of the sulphur are cleaved content from that couldthe surface provide by the th sulphydryle chlorine [9]. groups The for A-layer the covalent is thought anchorage to be ofinvolved the fatty because acids. of The the A-layer sulphur is content variable that in thickness could provide so if it the totally sulphydryl cleaved groups from the for exocuticle the covalent its contributionanchorage of to the the fatty epicuticle acids. thicknessThe A-layer from is wool variable would in bethickness of the order so if ofit 70totally nm [ 5cleaved]. Measurements from the ofexocuticle epicuticle its thicknesscontribution provided to the aepicuticle value of thickne 5 nm [12ss] from and given wool thatwould figure, be of only the order part of of the 70 A-layernm [5]. couldMeasurements be involved. of epicuticle The stability thickness of the provided epicuticle a proteinvalue of to 5 vigorousnm [12] and reagents given has that been figure, attributed only part to isopeptideof the A-layer cross-linking could be involved. [6]. This The is not stability necessarily of the epicuticle the case because protein theto vigorous inner root reagents sheath has and been the medullaattributed proteins to isopeptide have a high cross-linking isopeptide [6]. content This and is not yet arenecessarily degraded the by case proteases because to peptides the inner [18 –root20]. Moresheath likely, and the resistance medulla to degradationproteins have is thea high result isop ofeptide collaborative content forces and yet of hydrogen are degraded bonds, by disulphide proteases bondsto peptides and isopeptide [18–20]. More cross-links likely, in resistance a beta pleated to degradation sheet structure. is the result of collaborative forces of hydrogen bonds, disulphide bonds and isopeptide cross-links in a beta pleated sheet structure.

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Cosmetics 2019, 6, x FOR PEER REVIEW 5 of 8 4. The Cell Membrane Complex (CMC) Cosmetics 2019, 6, x FOR PEER REVIEW 5 of 8 4. The Cell Membrane Complex (CMC) A fascinating aspect of cuticle structure is the change that the plasma membranes between cuticle 4. TheA Cellfascinating Membrane aspect Complex of cuticle (CMC) structure is the change that the plasma membranes between cells (and cuticle-cortical) boundaries undergo in the development of the cuticle during growth of the cuticle cells (and cuticle-cortical) boundaries undergo in the development of the cuticle during hair. ThisA change fascinating has beenaspect the of subjectcuticle structure of extensive is the reports change and that reviews the plasma [21 ,22membranes]. Very little between is known growth of the hair. This change has been the subject of extensive reports and reviews [21,22]. Very cuticle cells (and cuticle-cortical) boundaries undergo in the development of the cuticle during of theirlittle formation is known andof their their formation composition and their and mostcomposition of our and knowledge most of our comes knowledge from TEM comes [22 ]from and also growth of the hair. This change has been the subject of extensive reports and reviews [21,22]. Very AFMTEM [23] studies[22] and ofalso keratinised AFM [23] hairstudies or wool.of keratinised The early hair cuticle or wool. intercellular The early cuticle membrane intercellular is a typical little is known of their formation and their composition and most of our knowledge comes from trilaminarmembrane unit membraneis a typical trilaminar consisting unit of amembrane bilipid layer consisting flanked of bya bilipid protein layer layers flanked and anby intercellularprotein TEM [22] and also AFM [23] studies of keratinised hair or wool. The early cuticle intercellular layers and an intercellular space between adjacent cells. However, at later stages of hair growth the spacemembrane between is adjacent a typical cells. trilaminar However, unit membrane at later stages consisting of hair of growtha bilipid thelayer separation flanked by of protein the nearly separation of the nearly keratinised cuticle cells involves a very different structure, the cell keratinisedlayers and cuticle an intercellular cells involves space a between very di adjacentfferentstructure, cells. However, the cell at later membrane stages of complex.hair growth A the central membrane complex. A central denser layer, the δ-layer, presumably protein, develops between denserseparation layer, the ofδ -layer,the nearly presumably keratinised protein, cuticle develops cells involves between a adjacentvery different cuticle structure, cells and isthe flanked cell by adjacent cuticle cells and is flanked by two low-density β-layers that are each interpreted to be a two low-densitymembrane complex.β-layers A that central are eachdenser interpreted layer, the toδ-layer, be a bilipidpresumably layer protein, (Figure 6develops). between bilipid layer (Figure 6). adjacent cuticle cells and is flanked by two low-density β-layers that are each interpreted to be a bilipid layer (Figure 6).

δ FigureFigure 6. The 6. The cell cell membrane membrane complex complex between between two cuticle cuticle cells cells showing showing the the central central dense dense δ—layer—layer and flankedand flanked by the by the two two low low density densityβ -layers.β-layers. FromFrom reference reference [24]. [24 ]. Figure 6. The cell membrane complex between two cuticle cells showing the central dense δ—layer and flanked by the two low density β-layers. From reference [24]. TEMTEM studies studies of theof the membrane membrane complex complex ofof the hair cuticle cuticle from from subjects subjects with with a mutation a mutation of the of the 18-MEA18-MEA synthetic synthetic pathway pathway (Maple (Maple Syrup Syrup Urine Urine disease disease (MSUD) (MSUD) [24 ][24] revealed revealed that that the likelythe likely position TEM studies of the membrane complex of the hair cuticle from subjects with a mutation of the position of the MEA is on only one face, the upper surface of each cuticle cell. It is this surface layer of the18-MEA MEA is synthetic on only onepathway face, the(Maple upper Syrup surface Urine of eachdisease cuticle (MSUD) cell. It[24] is thisrevealed surface that layer the oflikely 18-MEA of 18-MEA and other fatty acids that is responsible for the hydrophobic fibre surface (Figure 7). How and otherposition fatty of the acids MEA that is on is responsibleonly one face, for the the upper hydrophobic surface of each fibre cuti surfacecle cell. (Figure It is this7). surface How thelayer CMC the CMC cleaves to expose the underlying lipid has been proposed by Jones [6]. What properties the cleavesof 18-MEA to expose and the other underlying fatty acids lipidthat is has responsibl been proposede for the hydrophobic by Jones [6 ].fibre What surface properties (Figure 7). the How 18-MEA 18-MEA provide for hair is unclear but it has been suggested that since 18-MEA has the branched providethe CMC for hair cleaves is unclear to expose but the it underlying has been suggested lipid has been that proposed since 18-MEA by Jones has [6]. theWhat branched properties chain, the the chain, the melting point is much lower than that of the straight chain and as consequence the lipid melting18-MEA point provide is much for lower hair is than unclear that but of theit has straight been suggested chain and that as consequencesince 18-MEA thehas lipidthe branched layer on the layer on the cuticle surface would be more fluid. That could alter frictional properties. cuticlechain, surface the melting would bepoint more is much fluid. lower That than could that alter of the frictional straight properties.chain and as consequence the lipid layer on the cuticle surface would be more fluid. That could alter frictional properties.

Figure 7. The cell membrane complex in a Maple syrup disease hair showing the disruption of the β -layer on the upper surface of each cuticle cell (FCU). This demontrates that the stability of the layer is dependent upon the preence of 18-MEA. From reference [24]. Cosmetics 2019, 6, 32 6 of 8

5. Discussion: Some Unknown Aspects of Cuticle Structure that Need Innovative Techniques to Resolve The exocuticle: The formation of the droplets of exocuticle precursor, their fusion and migration to the outer surface are fascinating processes. Any study of this must also explain the distinct development of the A-layer with its significantly higher cystine content. The droplets appear to form by self-aggregation of the protein components. To study that possibility would require preparation of those proteins. Only KAP5 and KAP10 are known to be major components, so more protein analysis of the exocuticle is necessary to identify others. The gene sequences are known for KAP5 and KAP10 and could be prepared by expression in bacterial systems. Study of their interaction would then be amenable by standard proteomics methods such as those used in studying the binding of a KAP to an IF keratin [25]. The migration aspect however, is more difficult to study because it is a dynamic event. Could it be driven by a microtubule pathway? There is no report to indicate their involvement, so a thorough TEM study at many follicle levels using immunochemical tracking would be useful. The epicuticle Amino acid analyses have shown that there are cysteine residues to provide anchorage of fatty acids (the links are cleaved by the chlorination to elicit the epicuticle). As for the origin of the proteins, there is strong evidence indicating the involvement of the A-layer [26]. However it is possible that proteins from the cell membrane complex that compose the intercellular cement are also involved. In an effort to seek the proteins that have 18-MEA and other fatty acids attached by thioester bonds, experiments were conducted on fibres before keratinisation (G. Rogers and K. Koike, unpublished data). By using this approach, the keratin structures were not cross-linked and more amenable to studies in solution. The proteins such as the cell membrane complex were expected to be present in low abundance, so we faced a quantitative problem. Consequently, we used follicles epilated from sheepskin; thousands of follicles in anagen phase can be obtained. Their proteins were solubilised with 8M urea and the SH groups blocked with iodoacetamide. In extensive studies the authors used several different methods to identify any proteins with fatty acids covalently linked to cysteinyl groups. The most direct technique was to tag the acylated sites using acyl-biotin exchange [27]. This procedure entailed incubating the urea-soluble iodoacetamide-blocked proteins with hydroxylamine at 20 ◦C, which specifically breaks thioester linkages and releases the fatty acids. Any exposed SH groups of the protein were then tagged with a biotin moiety and any biotin- tagged proteins were captured with streptavidin beads. Unbound proteins were removed by washing and any bound proteins released from the beads. After concentrating the eluates, the proteins were examined by gel electrophoresis. The controls for the experiments were identical but with omission of the hydroxylamine step. After several attempts we could not obtain consistent results. We suspect that this was caused by an unexpected problem with the method. We found that the hydroxylamine in the presence of 8 M urea produced a gel that could have interrupted the hydrolysis. Gelation occurred in the absence of protein and no explanation could be found in the literature. These acyl-biotin exchange experiments need repeating but using other chaotropic agents to replace the urea, such as guanidine thiocyanate, or lithium salts, to avoid the gelation phenomenon but to keep the proteins in the solution.

6. At What Stage of Hair Growth is 18-MEA Attached to the Cuticle and How Does This Occur? The synthesis of 18-MEA occurs in the liver, it circulates in the blood and is available for capture and enzymic transfer to thiol groups presumably by a transferase enzyme. The known location of 18-MEA in hair is on the upper surface of the ﬂattened cuticle cells. This asymmetry as a structural feature of the β-layer and the question of how the presence of 18-MEA attached on one face only occurs is curious and remains without explanation. Cosmetics 2019, 6, 32 7 of 8

We don’t know at what follicle level 18-MEA is coupled to cuticle protein but it must occur when the proteins have sulphydryl groups available for acylation with fatty acids; the region defined as the elongation zone of the follicle [4] begins proximal to the hair bulb. One feasible method to locate these events would be to utilise TEM autoradiography. That would require injection of sufficient tritiated 18-MEA into a mouse or young guinea pig, preferably the latter because most of the follicles are in anagen. After selected times skin biopsies would be taken, processed, submitted to autoradiographic detection and examined in TEM. The problem here is that tritiated or C14-labelled 18-MEA are not commercially available. However, they could be synthesised for this purpose. Carbon-14 is usable for autoradiography at the TEM level but is less efficient than tritium.

7. Summary We now have a comprehensive view of the structure of the hair cuticle cell as a result of the application of an array of physical and chemical techniques. The power of TEM has played a central role because of the miniscule nature of the hair follicle and the macromolecular components of the hair. Some unanswered questions have been discussed in this essay and clearly they are of fundamental interest but they will be diﬃcult to solve and undoubtedly will require novel approaches. To justify future research in that direction will probably largely depend on the applicability of further knowledge to the usage and treatment of hair.

Funding: The preparation of this review received no external funding. The author acknowledges the provision of writing facilities by the Department of Molecular and Biomedical Science. Conﬂicts of Interest: The author declares no conﬂict of interest.

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