Analysis of two pore channel proteins in Dictyostelium development

Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular responses including development and proliferation. Calcium (Ca2+) is stored in both neutral and acidic stores and its release through gated channels has been implicated in regulating development in Dictyostelium discoideum. This thesis aims to understand the roles of the calcium channel proteins, in particular the two-pore channel proteins (TPCs), found on acidic stores in Ca2+ signalling during Dictyostelium development. Bioinformatic analysis indicates conservation of a gene encoding an orthologue of TPC2 in Dictyostelia and, similar to plant TPCs, a Ca2+ sensing domain is predicted along with a novel potential calmodulin binding site. To investigate the role of intracellular Ca2+ channels, a series of strains was generated, disrupted in one or more of genes encoding the channels TPC2 and mucolipin (TRP-ML), predicted to be located on acidic stores, and IplA, located on neutral stores. All disrupted strains, including one lacking all three channels, are able to complete development. However, strains lacking TPC2 show a pronounced delay in early development, correlating with reduced expression of some early developmental genes. Vesicles derived from tpc2-null cells show normal Ca2+ release compared to parental cells but an increased rate of Ca2+ uptake. During early development, the pH of acidic vesicles is increased in the absence of TPC2. However development of tpc2-null cells showed increased sensitivity to weak bases in producing fewer aggregates but resistance to sodium chloride and weak bases in later development suggesting a complex role for TPC during development. In vivo cytosolic Ca2+ responses were analysed in strains expressing an ultra-sensitive Ca2+ indicator YC-Nano 15. Growing tpc2- and iplA- cells have lower basal cytosolic Ca2+ than parental Ax2 cells. Intercellular Ca2+ waves were observed in aggregates from Ax2, mcln- and tpc2- cells but were greatly reduced in iplA- aggregates, as was the increase in cytosolic Ca2+ in response to extracellular cAMP. In tpc2- aggregates, wave frequencies were reduced and the response to cAMP addition abolished after treatment with caffeine, a known adenylyl cyclase inhibitor in Dictyostelium. This work demonstrates that TPC2 plays a role in the early stages of Dictyostelium development. TPC2 is important for pH regulation in acidic vesicles and cytosolic Ca2+ levels, either or both of which could influence development either directly or via changes in early developmental gene expression.