Background: Microtubule associated proteins are a group of proteins well characterised for their role in the stabilisation and polymerisation of microtubules, but they have been increasingly demonstrated to have a more diverse role within cells. Tau is a microtubule associated protein, well studied for its pathological role in Alzheimer’s disease. Recent studies have provided evidence that tau is also expressed in the epidermal compartment of the skin. However, the precise roles of tau during epidermal stratification and differentiation are currently unknown. Aims: This study aims to investigate the role of tau in epidermal stratification and differentiation and its function in cutaneous squamous cell carcinoma (cSCC). Methods: Immunostaining was used to investigate tau expression in clinical samples, 2D cultures and 3D skin equivalents. To explore the functional significance of tau during keratinocyte differentiation and stratification, siRNA and shRNA were used to downregulate tau expression in keratinocytes and the cSCC cell line, A431. Gene and protein expression were assessed using RT q-PCR and Western blotting, and changes in cytoskeletal organisation were determined using immunofluorescence. The overexpression of tau was conducted through the generation of stable cell lines with a doxycycline inducible overexpression of tau. The functional significance of tau overexpression was determined using a combination of RT q-PCR, Western blot and immunofluorescence analyses of 2D epidermal keratinocyte culture, MatTek© 3D epidermal models and cSCC cells in vitro. Co-Immunoprecipitation (CoIP) and subsequent proteomic analysis were performed to identify tau binding partners in growing and differentiated keratinocytes. Results: In healthy human skin, tau expression was upregulated in the differentiated population of suprabasal keratinocytes and displayed a distinct isoform specific expression throughout the epidermis. Overexpression of tau in vitro was found to initiate keratinocyte differentiation in 2D culture and 3D epidermal models. CoIP and subsequent proteomic analysis identified a discrete pattern of tau interactions in keratinocytes cultured under growing and differentiated conditions, with a specific subset of nuclear, cytoplasmic and membrane associated proteins identified to interact with tau through different stages of keratinocyte differentiation. Finally, in cSCC tau expression was linked to the differentiation status of the tumours. Furthermore, the downregulation and overexpression of tau was sufficient to induce changes to cytoskeletal organisation and cellular differentiation in cSCC in vitro. Conclusions: This study revealed that tau can orchestrate epidermal cell fate, differentiation and stratification in healthy human skin and cSCC. These findings are therefore anticipated to be a starting point for more functional research into the binding partners of tau during epidermal stratification, differentiation and their subsequent function in cancer.

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