Macrofibrils, the main structural features within the cortical cells of mammalian hair shafts, are long composite bundles of keratin intermediate filaments (KIFs) embedded in a matrix of keratin-associated proteins. The KIFs can be helically arranged around the macrofibril central axis, making a cylinder within which KIF helical angle relative to macrofibril axis increases approximately linearly from macrofibril centre to edge. Mesophase-based self-assembly has been implicated in the early formation of macrofibrils, which first appear as liquid-crystal tactoids in the bulb of hair follicles. Formation appears to be driven initially by interactions between pre-keratinized KIFs. Differences in the nature of these KIF-KIF interactions could result in all macrofibrils being internally twisted in a single handedness, or a 50:50 mixture of handedness within each cortical cell. We data-mined 41 electron tomograms containing three-dimensional macrofibril data from previously published studies of hair and wool. In all 644 macrofibrils examined we found that within each tomogram all macrofibrils had the same handedness. We concluded that earlier reports of left- and right-handed macrofibrils were due to artefacts of imaging or data processing. A handedness marker was used to confirm (using re-imaged sections from earlier studies) that, in both human and sheep, all macrofibrils are left-handed around the macrofibril axis. We conclude that this state is universal within mammalian hair. This also supports the conclusion that the origin of macrofibril twist is the expression of chiral twisting forces between adjacent KIFs, rather than mesophase splay and bending forces relaxing to twisting forces acting within a confined space.
- MeSH
- Cytoskeleton chemistry ultrastructure MeSH
- Intermediate Filaments chemistry ultrastructure MeSH
- Keratins chemistry ultrastructure MeSH
- Humans MeSH
- Sheep genetics MeSH
- Electron Microscope Tomography MeSH
- Hair chemistry ultrastructure MeSH
- Wool chemistry ultrastructure MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Epidermolysis bullosa simplex (EBS) is an inherited skin disorder caused by mutations in the keratin 5 (KRT5) and keratin 14 (KRT14) genes, with fragility of basal keratinocytes leading to epidermal cytolysis and blistering. OBJECTIVES: In this study, we characterized mutations in KRT5 and KRT14 genes in patients with EBS and investigated their possible structure-function correlations. MATERIALS AND METHODS: Mutations were characterized using polymerase chain reaction (PCR) and DNA sequencing. Further, to explore possible correlations with function, the structural effects of the mutations in segment 2B of KRT5 and KRT14 and associated with EBS in our patients, as well as those reported previously, were modelled by molecular dynamics with the aid of the known crystal structure of the analogous segment of human vimentin. RESULTS: We have identified mutations in the KRT5 and KRT14 genes in 16 of 23 families affected by EBS in the Czech Republic. Eleven different sequence variants were found, of which four have not been reported previously. Novel mutations were found in two patients with the EBS-Dowling-Meara variant (EBS-DM) [KRT14-p.Ser128Pro and KRT14-p.Gln374_Leu387dup(14)] and in three patients with localized EBS (KRT14-p.Leu136Pro and KRT5-p.Val143Ala). Molecular dynamics studies show that the mutations p.Glu411del and p.Ile467Thr perturb the secondary alpha-helical structure of the mutated polypeptide chain, the deletion p.Glu411del in KRT14 has a strong but only local influence on the secondary structure of KRT14, and the structural impact of the mutation p.Ile467Thr in KRT5 is spread along the helix to the C-terminus. In all the other point mutations studied, the direct structural impact was significantly weaker and did not destroy the alpha-helical pattern of the secondary protein structure. The changes of 3-D structure of the KRT5/KRT14 dimer induced by the steric structural impact of the single point mutations, and the resulting altered inter- and intramolecular contacts, are spread along the protein helices to the protein C-terminus, but the overall alpha-helical character of the secondary structure is not destroyed and the atomic displacements induced by mutations cause only limited-scale changes of the quaternary structure of the dimer. CONCLUSIONS: The results of molecular modelling show relationships between patients' phenotypes and the structural effects of individual mutations.
- MeSH
- Child MeSH
- Adult MeSH
- Epidermolysis Bullosa Simplex genetics pathology MeSH
- Phenotype MeSH
- Microscopy, Fluorescence MeSH
- Genetic Predisposition to Disease MeSH
- Intermediate Filaments ultrastructure MeSH
- Keratin-14 genetics MeSH
- Keratin-5 genetics MeSH
- Skin ultrastructure MeSH
- Humans MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Child, Preschool MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The collapse of vimentin caused by some xenobiotics correlates with the loss of structural integrity of the seminiferous epithelium. In this study, we investigated the effect of busulphan (an anticancer drug with toxic effects on dividing germ cells) on vimentin filament distribution in rat seminiferous epithelium and compared it with changes found in testes of unilaterally cryptorchid rats. In the seminiferous epithelium, the vimentin labelling was observed only in the Sertoli cells, showing a stage-specific arrangement of the filaments. Both busulphan treatment and cryptorchism caused altered distribution of vimentin filaments in the Sertoli cells. In both models, the apical vimentin filaments collapsed towards the nuclei and were disorganized in the basal region of the Sertoli cells while the germ cells were diminished in the epithelium. After the busulphan effect subsided (4 weeks after administration), spermatogenesis began to restore and vimentin filaments began to organize in basal and perinuclear regions of Sertoli cells among the spermatogonia and spermatocytes. Vimentin labelling of the sloughed material in the lumen of cryptorchid testes (but not in busulphan treated animals) was observed. We conclude that the Sertoli cell vimentin filaments play an important role in the maintenance of spermatogenesis, their damage is associated with the seminiferous epithelium disintegration and their restoration with a recovery of spermatogenesis after the unfavourable conditions subside.
- MeSH
- Busulfan pharmacology MeSH
- Time Factors MeSH
- Financing, Organized MeSH
- Immunohistochemistry MeSH
- Intermediate Filaments chemistry metabolism ultrastructure MeSH
- Rats MeSH
- Rats, Wistar MeSH
- Seminiferous Epithelium metabolism ultrastructure MeSH
- Spermatogenesis physiology genetics drug effects MeSH
- Testis metabolism ultrastructure MeSH
- Organ Size MeSH
- Vimentin biosynthesis metabolism drug effects MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- MeSH
- Axons pathology ultrastructure MeSH
- Diabetes Mellitus, Experimental chemically induced pathology MeSH
- Intermediate Filaments ultrastructure MeSH
- Rats MeSH
- Neurons, Afferent ultrastructure MeSH
- Peripheral Nerves pathology MeSH
- Nerve Regeneration MeSH
- Schwann Cells ultrastructure MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
