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Title: Function of the laminin-derived protein LaNt α31 in corneal epithelium
Author: Iorio, Valentina
ISNI:       0000 0004 6496 3754
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Laminin N-terminus α31 (LaNt α31), a member of the relatively new LaNt protein family, is a product of alternative splicing from the laminin (LM) α3 encoding gene (LAMA3). Prior to these studies, very little was known about LaNt α31 biology; it had been shown to co-localise with LM α3β3γ2 (LM332) in the basement membrane (BM) of the skin and to play a role in adhesion and migration of epidermal cells. Still, no direct insight into the mechanism behind this effect has been described. In this study, we performed the first analysis of LaNt α31 in the eye. Immunohistochemistry reveals that the protein is differentially distributed across the regions of the epithelium in the anterior surface of the eye. Specifically, LaNt α31 localises intracellularly through all layers of the corneal epithelium, but is restricted to the basal layers of the limbus and conjunctiva. Hence, we sought to investigate about LaNt α31 functional roles in corneal epithelial cells (hTCEpi) and its interplay with LM during corneal epithelial matrix assembly. Our functional studies demonstrate that knockdown of intracellular LaNt α31 has no discernible effect on hTCEpi, in which LaNt α31 is not normally matrix-associated. However, when overexpressed by adenoviral delivery, LaNt α31 is deposited into the extracellular matrix (ECM) and causes a number of phenotypic effects including; impaired cell adhesion to LM111, changes to LM organisation, increased cell spreading, early recruitment of Collagen (Col) type XVII to β4 integrin in hemidesmosome (HD) like-complexes and mislocalisation of focal adhesions (FAs). Increased LaNt α31 expression also leads to significantly decreased motility at times of new matrix synthesis, with this migration defect rescued when hTCEpi are provided with a cell-derived preformed ECM. Live imaging experiments indicate that GFP-tagged LaNt α31 clusters at the periphery of cells, where it co-distributes and is deposited along with LM β3. As cells move across a preformed matrix, clusters of LaNt α31 rapidly form and dissociate intracellularly, alongside LM deposits. To study the effects of overexpression in-vivo, we generated a human keratin 14 (hK14) promoter-driven LaNt α31 overexpressing mouse model by embryo microinjection. Using this construct, we obtained far fewer offspring than would normally be expected and were not able to detect expression of the protein product from the transgene in the animals carrying the construct. Based on these data, we hypothesise that, in this model system, LaNt α31 overexpression has induced lethality. Together, these data demonstrate that LaNt α31 influences the assembly of LM matrix in hTCEpi affecting fundamental cell functions, and implicate the LaNt proteins as regulators of other LM dependent processes including wound repair, differentiation and tissue homeostasis.
Supervisor: Hamill, K. J. ; Sheridan, C. ; Bou-Gharios, G. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral