In vivo formation steps of the hard α-keratin intermediate filament along a hair follicle: Evidence for structural polymorphism (original) (raw)
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The Intermediate Filament Architecture as Determined by X-Ray Diffraction Modeling of Hard α-Keratin
Biophysical Journal, 2004
Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard a-keratin intermediate filaments under in vivo conditions. In short, the a-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.
The intermediate filament structure of human hair
Biochimica et Biophysica Acta (BBA) - General Subjects, 1995
X-ray diffraction studies of hard a-keratin have led to a proposed model lbr the lateral arrangement of molecules within the keratin fibrils of tissues such as hair, nail and claw. Using low-angle synchrotron radiation to examine human scalp hair we have obtained discrete equatorial diffraction maxima which have not been reported previously. These reflections can be divided into three subsets. The first of these reveals the information that the hair fibres consist basically of cylindrical fibrils arranged in a disordered lattice. The mean diameters of these cylinders have been determined, together with their average separation. The diameters of the protofibrils have been determined from the second set. The third set, a set of diffuse arcs, index onto a spacing which is characteristic of the disordered components of the matrix.
Biophysical journal, 2004
Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard alpha-keratin intermediate filaments under in viv...
The Journal of Cell Biology, 1983
We have used scanning transmission electron microscopy to elucidate the question of how intermediate filament (IF) subunits of widely differing mass can all form morphologically similar IF. From scanning transmission electron micrographs, the distributions of mass were determined for three types of epidermal keratin IF reassembled in vitro from mixtures of subunits with substantially different masses, viz., "light" and "heavy" human keratins with [Mr] = 50,000 and 56,000, respectively, and mouse keratins of [Mr] = 63,000. Their principal assembly products were found to average 22, 25, and 29 kdalton/nm, respectively. These densities, which correspond to immature "minimal form" IF (Steven, A. C., J. Wall, J. Hainfeld, and P. M. Steinert, 1982, Proc. Natl. Acad. Sci. USA., 79:3101-3105), are directly proportional to the average subunit masses. The human keratin IF (but not those of mouse) also contained minor amounts (15-20%) of more massive polymers aver...
Elucidating the early stages of keratin filament assembly
The Journal of Cell Biology, 1990
Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed human K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel. 1. Abbreviations used in this paper: 1B, inclusion body; IBF, IB-rich fraction; IE intermediate filament. formed with pETK14; (lane 10) IBF from bacterial extract in lane 9; (lane 11) Mono Q purification of K14 IBF in lane 10; and (lane 12) Superose 12 purification of pooled Mono Q fractions from lane 11. Molecular masses of epidermal keratins are indicated at left.
Biochemistry Research International, 2011
High-and low-angle X-ray diffraction studies of hard α-keratin have been studied, and various models have been proposed over the last 70 years. Most of these studies have been confined to one or two forms of alpha keratin. This high-and low-angle synchrotron fibre diffraction study extends the study to cover all available data for all known forms of hard α-keratin including hairs, fingernails, hooves, horn, and quills from mammals, marsupials, and a monotreme, and it confirms that the model proposed is universally acceptable for all mammals. A complete Bragg analysis of the meridional diffraction patterns, including multiple-time exposures to verify any weak reflections, verified the existence of a superlattice consisting of two infinite lattices and three finite lattices. An analysis of the equatorial patterns establishes the radii of the oligomeric levels of dimers, tetramers, and intermediate filaments (IFs) together with the centre to centre distance for the IFs, thus confirming the proposed helices within helices molecular architecture for hard α-keratin. The results verify that the structure proposed by Feughelman and James meets the criteria for a valid α-keratin structure.
The complex mechanical properties of biomaterials such as hair, horn, skin, or bone are determined by the architecture of the underlying fibrous bionetworks. Although much is known about the influence of the cytoskeleton on the mechanics of isolated cells, this has been less studied in tridimensional tissues. We used the hair follicle as a model to link changes in the keratin network composition and architecture to the mechanical properties of the nascent hair. We show using atomic force microscopy that the soft keratinocyte matrix at the base of the follicle stiffens by a factor of ∼360, from 30 kPa to 11 MPa along the first millimeter of the follicle. The early mechanical stiffening is con-comitant to an increase in diameter of the keratin macrofibrils, their continuous compaction, and increasingly parallel orientation. The related stiffening of the material follows a power law, typical of the mechanics of nonthermal bending-dominated fiber networks. In addition, we used X-ray diffraction to monitor changes in the (supra)molecular organization within the keratin fibers. At later keratinization stages, the inner mechanical properties of the macrofibrils dominate the stiffening due to the progressive setting up of the cys-tine network. Our findings corroborate existing models on the sequence of biological and structural events during hair keratinization.
Unzipping the cuticle of the human hair shaft to obtain micron/nano keratin filaments
Biopolymers, 2006
An attempt has been made to obtain intact individual keratin filaments of various levels from micron cortical, micron macrofibril to nano intermediate filament and polypeptide α-helix from the human hair shaft. The feasibility of this initiative has been largely demonstrated by finding that there is a longitudinal seam/zipper on the cuticle of the human hair shaft, which can be unzipped by certain solvents such as performic acid and urea, allowing one to use an anatomical approach to separate intact individual micron/nano filaments. Micron cortical and macrofibril filaments have been obtained. It is also found that the cortical filaments are twisted together to form a yarn, giving rise to the strength for the hair shaft; and that individual cortical filaments are often 2-2 paired in a similar structure to the double helix. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 614–618, 2006This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
BIOCELL
The differentiation of cells composing mature human hairs produces layers with different corneous characteristics that would tend to flake away one from another, as in the corneous layer of the epidermis, without anchoring junctions. It is likely that cell junctions established in the forming cells of the hair bulb are not completely degraded like in the corneous layer of the epidermis but instead remain in the hair shaft to bind mature cuticle, cortex, and medulla cells into a compact hair shaft. During cell differentiation in hairs, cell junctions seem to disappear, and little is known about the fate of junctional proteins present in the mature human hair shaft. The present ultrastructural immunogold study has detected some marker proteins of adhesion junction (cadherin and beta-catenin) and tight junctions (occludin and cingulin) that are still present in cornified hairs where numerous isopeptide bonds are detected, especially in the medulla. This qualitative ultrastructural study indicates that aside from the cell membrane complex, a long corneo-desmosome bonding cortex and cuticle cells, also sparse adherens and tight junction remnants are present. It is suggested that the cornification of these junctions with the incorporation of their proteins within the mature corneous material of the hair shaft likely contributes to maintaining the integrity of the mature hair. This information will also allow us to evaluate the effects of different chemical components present in hair formulations and stains on these junctional proteins and the consequent integrity of the hair shaft.
Growth of human hair follicle keratinocytes in vitroUltrastructural features of a new model
Journal of The American Academy of Dermatology, 1987
A simple experimental technique was developed to provide an in vitro model for the study of human follicular keratinocytes. Anagen-phase human hairs were plucked from the scalp of healthy individuals; the follicles were separated, plated on coverslips coated with collagen G, and cultivated in McCoy 5A Medium in a CO2-incubator at 37 ~ C. Light and electron microscopy after 1, 2, 3, and 6 weeks showed selective and progressive cell growth with keratinocyte differentiation, producing multilayered cultures of cells joined with fully developed desmosomes. Three distinct patterns of differentiation, leading to the formation of an incomplete horny layer, were seen. The particular arrangement of tonofilaments, the considerable amounts of cytoplasmic glycogen, and the absence of malpighian differentiation were ultrastructural indicators of the follicular origin of the cultured cell population, which most likely grew from the outer root sheath of the hair. This technique may provide a promising model on which to base further studies of hair biologic processes and hair growth. (J AM ACAD DERMATOL 1987;17:779-86.) The hair bulb is a complex cutaneous appendage with a dynamic structure, as related to its life cycle. Its periodic growth occurs under the influence of numerous endogenous and environmental factors. The interconnection between these factors represents a true challenge when hair researchers attempt to evaluate individual experimental findings such as the effect of given substances on the biologic processes and growth of hair. In particular, human hair growth has always been a difficult field of investigation, since experimental procedures were restricted by the lack of corresponding