title: On the Development of Recombinant Ca2+ Indicators for the Measurement of Extracellular Ca2+ with a Focus in the Epidermis
Creators: name: Purohoo, Muhammad Saajid
Publisher: name: University of Liverpool Repository
Year: 2025

description: The Calcium ion (Ca2+) is a secondary messenger regulating many key processes within cells. Intracellular Ca2+ signalling is regulated by numerous Ca2+ channels, pumps and exchangers located on intracellular organelles in cells and on the PM to fine tune the signal to bring about specific downstream processes. Also important is extracellular Ca2+ (Ca2+e) which regulates other cellular and tissue processes in many systems. Ca2+ is extruded into the extracellular space to terminate signals and prevent cell toxicity, and extruded Ca2+ forms microdomains capable of eliciting downstream signals on their own, where Ca2+ acts as a primary messenger. Ca2+e as a primary messenger is vital especially in cells where the Calcium Sensing Receptor (CaR) is expressed. In the epidermis, a Ca2+ gradient which drives cell and tissue development exists and is maintained by active cycling between cells in the different layers. In low Ca2+, keratinocytes proliferate in the basal layer and differentiate as they move into the higher layers where Ca2+ is high. So far, the Ca2+ switch altering keratinocyte development has not yet been identified. Furthermore, disruption of the permeable barrier in the epidermis leads to loss of the Ca2+ gradient. Work into improving barrier repair has revealed accelerated barrier recovery can be both induced and inhibited by elevated intracellular Ca2+. Current Ca2+ measuring techniques have inherent limitations and barriers posed by cells and tissues themselves. Consequently, better tools are needed to provide a better spatiotemporal insight into Ca2+ transactions in tissues where extracellular Ca2+ drives development. In this thesis, three different PM-bound probes for measuring [Ca2+]e were characterised and developed to allow for investigation in the epidermis and other tissues. First, RCEPIA1pm, previously designed and to be targeted to the extracellular environment on the PM, was characterised in varying [Ca2+]e and along with the dye Fluo-8 in the presence of Ca2+ mobilising agonists to simultaneously measure cytoplasmic Ca2+ (Ca2+c) and Ca2+e. Our data showed that RCEPIA1pm successfully senses changes in Ca2+e and can be used simultaneously with cytoplasmic dyes. Clones generated from RCEPIA1pm can be used in 3D tissue environments to better understand Ca2+ transactions. A bicistronic plasmid encoding a green PM bound probe, GCEPIA1pm, and a red reference signal, lssmCherry1, joined by a 2A peptide, was then designed as a ratiometric solution to address issues usually encountered when using single wavelength indicators. Our data showed successful targeting of GCEPIA1pm to the PM and lssmCherry1 to the cytoplasm Ca2+-sensing by GCEPIA1pm and the potential for ratiometric measurements. To simultaneously measure intracellular and extracellular Ca2+ dynamics, a third bicistronic plasmid with two Ca2+ sensing fluorophores, GCaMP6210pm for Ca2+e sensing and REXGECO1 for ratiometric Ca2+c measurements, was designed. Although successful localised expression was obtained, GCaMP6210pm did not display desirable Ca2+e sensing properties while REXGECO1 was successful at detecting Ca2+c elevations and decreases. Through changing GCaMP6210pm with another Ca2+ sensor with more desirable properties, a plasmid that will allow spatiotemporal measurement of intracellular and extracellular Ca2+ dynamics is feasible. Current Ca2+ measurement systems do not possess the ability to provide spatiotemporal measurements of cellular Ca2+ dynamics. In this thesis, we successfully developed probes capable of measuring Ca2+e and simultaneous intracellular and extracellular Ca2+, which can then be used in 3D tissues through generation of clones and other expression systems to study the role Ca2+ plays in tissue development.