The development of new molecular tools to investigate epidermal Ca2+ homeostasis

Talbot, WJ (2018) The development of new molecular tools to investigate epidermal Ca2+ homeostasis. PhD thesis, University of Liverpool.

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Abstract

The calcium ion (Ca2+) is a ubiquitous second messenger with well-established roles across many tissue systems. In the epidermis, Ca2+ has long been identified as a primary regulator of proliferation, differentiation, and barrier homeostasis. A gradient in Ca2+ has been observed with the general view that both intracellular and extracellular Ca2+ concentrations are lowest in the basal layer and peaking in the upper granular layer. Work in vitro has identified that modifications of extracellular Ca2+ dictate the proliferation state of keratinocytes. However, this extracellular switch has not been elucidated in vivo. In addition, despite defective Ca2+ uptake into organelles such as ER and golgi being shown to disrupt barrier homeostasis, only now is it emerging that mitochondria may play a significant role in epidermal homeostasis on regulation and differentiation as well as apoptosis. In this thesis, a bi-fold approach was taken in order to develop new molecular tools in order to allow the investigation into the role of extracellular Ca2+ in epidermis in vivo and the role of mitochondrial Ca2+ in proliferating and differentiating HaCaT keratinocytes. Firstly, two extracellular genetically encoded Ca2+ indicators targeted to the extracellular surface were designed and generated. The first CatchER, was unsuccessful despite protein production being confirmed. The second, green and red (G/R)-CEPIA1pm, was successfully targeted to the plasma membrane and using live cell imaging, Ca2+ sensitivity of the probe was confirmed. Incorporation of this novel probe into 3D-culture systems has the potential to throw new light on the role of extracellular Ca2+ in tissues. In order to investigate the role of mitochondrial Ca2+ on keratinocyte homeostasis, the recently described mitochondrial Ca2+ uniporter was knocked out using the CRISPR/Cas9 gene editing system. Using qPCR and Western blotting, the relative expression of some MCU family proteins was investigated. Mitochondrial aequorin was used to investigate Ca2+ handling in keratinocytes in proliferating or differentiating conditions. In addition, in the same conditions, proliferation studies were used in order to assess the effect of reduced mitochondrial Ca2+ uptake on keratinocyte homeostasis. HaCaT keratinocytes grown in low Ca2+ proliferate at a higher rate than those in high Ca2+ conditions. This increased growth rate is accompanied by an increase in agonist-evoked mitochondrial Ca2+ uptake and also a decrease in the gene expression of the MCU inhibitor, MICU2. HaCaT keratinocytes devoid of the MCU protein struggle to proliferate in high Ca2+ conditions, with cell death and cell senescence appearing to occur. This reduction in proliferation rate of MCU -/- HaCaT keratinocytes is partly rescued when grown in low Ca2+ conditions. These results indicate an important role for mitochondrial Ca2+ in keratinocyte and in turn, epidermal homeostasis.

Item Type:	Thesis (PhD)
Divisions:	Faculty of Health and Life Sciences > Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	15 Aug 2018 07:32
Last Modified:	19 Jan 2023 01:31
DOI:	10.17638/03022782
Supervisors:	Simpson, Alec Tepikin, Alexei Rizzuto, Rosario
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3022782