An Investigation of Hair and its Keratin Associated Proteins using Advanced Light Microscopy

Abstract

Quantification and understanding of hair damage are often derived using semi-quantitative means which provide limited information regarding the whole hair. Furthermore, the roles of the varied keratin associated proteins (KAPs), which are the second most abundant proteins within hair, are poorly understood. Finally, the movement of compounds into the hair fibre has never previously been analysed dynamically or using the range of techniques currently available.
Fluorescence lifetime imaging microscopy (FLIM) and correlative techniques are utilised to quantify and understand the chemical changes taking place following damage. Super-resolution imaging techniques, including Airyscan and 3D structured illumination microscopy (3D-SIM) are used for the imaging of a selection of fluorescently-tagged KAPs within transiently transfected HaCaT cells in order to describe their cell biology. Fluorescence recovery after photobleaching (FRAP) is also applied to gather information regarding the dynamics of these proteins. Additionally, Airyscan and FRAP are utilised for the analysis of fluorescent dye movement within hair to characterise dye pathways and partitioning in hair dynamically.
FLIM provides a rapid and sensitive means to quantify oxidative damage to hair spatially, which is correlated to the conversion of tryptophan, through oxidation, to products including kynurenine. The uptake of dye into hair is dependent upon the size and lipophilicity of the molecule and may be limited due to a cuticle-cortex boundary observed. Knowledge of compound uptake into hair may be translated for the development of future cosmetics. The KAP subfamilies display distinct subcellular localisations to cytoskeletal components including epidermal keratins and actin structures. Functional characterisation of KAPs carried out in this study could allow a targeted approach to KAP-KAP and KAP-keratin interactions in future cosmetic treatments. This understanding may also be of relevance to the mechanism of certain diseases unrelated to the hair follicle, and indeed for the production of keratin-based biomaterials.