Calcium signalling and calcium pools in a Drosophila epithelium

Southall, Tony David (2003) Calcium signalling and calcium pools in a Drosophila epithelium. PhD thesis, University of Glasgow.

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The Drosophila Malpighian tubule is an ideal model epithelium for the study of fluidtransport and cell signalling. The tubule is the primary osmoregulatory tissue in the fly and Ca2+ signalling plays a critical role in controlling the fluid secretion rate of this organ. This project describes the use of powerful transgenic and fluorescent reporter techniques to further understand the mechanics of Ca2+ signalling in the cells of this tissue. In particular,the contribution of internal Ca2+ stores to the diuretic peptide-induced Ca signals has been investigated. In an attempt to unravel the role of the ER in these signals, an approach involving the Ca2+ sensitive photoprotein aequorin was undertaken. Transgenic flies were generated that could express ER-targeted aequorin; this in theory would have allowed real-time measurements of [Ca2+] ER in tubules during agonist stimulation. Unfortunately this method is presently not achievable in Drosophila, due to problems with targeting signals and the retention properties of the insect ER. As an alternative strategy a fluorescent Ca2+ reporter was developed that is targeted and functional in the ER. Transgenic flies were generated that could express this new reporter, ERpicam, in a cell specific manner. This has allowed realtime monitoring of [Ca2+]ER levels in a live intact tissue during stimulation with neuropeptides. The results were surprising, as they imply that the ER plays no role in the generation of LP3-induced Ca2+ signals in some cells of the tubule. The impact of these Ca2+ signals on mitochondrial Ca2+ levels was also investigated using targeted aequorin and new improved targeted fluorescent reporters. Mitochondria in the tubule do take up Ca2+ during the signalling events, however, the dynamics of this uptake are in contrast with the majority of data collected in other cell systems. Both the aequorin and fluorescent reporter techniques revealed that [Ca2+]mt levels in both cell types of the tubule do not increase in conjunction with the primary IP3-induced component of the signal but instead with the slower secondary response. This project also describes the identification and characterisation of the Drosophila secretory pathway Ca2+/Mn2+ ATPase (SPCA). The discovery that this multiply-sphced ATPase is targeted to multiple organelles will have important implications for further understanding of Ca2+ and Mn2+ transport in all cellular systems. Furthermore, it has uncovered roles for this protein in tubule function and has proved to be a powerful tool for dissecting the Ca2+ signals in this tissue through overexpression studies. Most interestingly, overexpression of a Golgi-localised SPCA results in a potentiation of EP3-mduced Ca increases in cells of the tubule, implying that this organelle plays a significant role in generating these responses. During the initial investigation of the Drosophila SPCA, development of a new gene knock-in method for Drosophila was theorised in an attempt to elucidate the expression and function of the SPCA. This technology is based on spliceosome-mediated trans-splicing. It can allow specific targeting of pre-mRNAs in transgenic Drosophila and enable reprogramming of the mature mRNA. The system has been adapted and successfully demonstrated in transgenic flies, however further work and refinement is needed before it can be used as a generic tool. These approaches have made significant advances into understanding the unusual Ca2+ signalling events of this epithelium and this work can be continued to gain further insight into how renal function is controlled in Drosophila and higher organisms.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Shireen Davies
Keywords: Physiology
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-74205
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Sep 2019 15:33
Last Modified: 23 Sep 2019 15:33

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