A combination of microscopic techniques to assess the self-assembly of keratins in a developing wool fibre

Produced through sustainable, simple and cheap methods, keratin is one of the naturally-derived biomaterials frequently used in the biomedical and pharmaceutical industries. Characteristic threadlike outgrowths of the outer layer of the skin (epidermis) that form the fur, hair, or wool of a mammal contain 95% keratin which develops from the follicle. Keratins are fibrous insoluble proteins belonging to the intermediate filament superfamily and based on their molecular weight and acidity, they are classified as Type I and Type II keratins. During hair morphogenesis, heterodimers of keratins associate to form tetramers which further polymerize to form unit-length filaments (ULFs), which are the monomer of keratin intermediate filament (KIFs). These KIFs mature into a fibre through either self-assembly or extensive cross-linking with associated proteins, neither of which are well understood. A combination of TEM methods has contributed immensely to current existing knowledge on keratin assembly in hair. Artefacts arising from sample processing including fixation, dehydration, embedding, sectioning and staining reduce the resolution power of TEM from atomic scales to nanometres. Cryofixation by high-pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. As the first aim of this thesis, the feasibility of replacing chemical conventional method with HPF and dehydration with FS for preserving the ultrastructure and antigenic epitopes in developing wool fibres was investigated. The ultrastructure of keratin and cellular components in a developing wool fibre were observed to be preserved in a close-to- native state. In addition to sample processing for TEM, the fidelity of the structural studies is critically dependent on minimising biological changes that may occur between follicle biopsy and TEM processing. Holding solutions play a key role to maintain follicle viability and function. HPF-FS enabled to assess the impact of selected holding solution on the wool follicle ultrastructure. The second aim of the thesis, enabled by the methods developed in the first part, was to characterize the interaction between hair keratins in the cortex of growing hairs. Since the antigenic epitopes were also well-preserved with HPF-FS, the distribution and interaction of first keratins of the hair cortex, Type I K35, Type II K85 and Type I K31, were investigated. Correlative light and electron microscopy in combination with darkfield TEM indicated that the initial keratin filament bundles in the early fibre cortex to be mainly composed of heterodimers of K31 and K85. In addition to keratins, increased distribution of a keratin-associated protein (KAP16.1) in a mutant variant was also established. Overall, HPF-FS was shown to be an excellent alternative to preserve both the ultrastructure and antigenic epitopes in wool follicles. Combined with dark field TEM, the organization of different keratins and KAPs in a developing fibre can easily be determined