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The aim of this study was to investigate the role of Ca²⁺ signalling pathways in the growth, morphogenesis and hyphal reorientation responses of <i>C. albicans</i>. The genes <i>CCH1</i> and <i>MID1</i> were identified in <i>S. cerevisiae</i> as encoding putative Ca²⁺ channels. These genes have since been shown to compose a Ca²⁺ channel complex. Homologues of these genes were identified and cloned from <i>C. albicans</i>. <i>CaMID1</i> was disrupted by the Ura-blaster method, and the resulting mutant characterised. The <i>C. albicans</i> <i>mid1</i> mutant strain was sensitive to the depletion of Ca²⁺ the presence of cell wall perturbing compounds such as SDS and Calcofluor. It formed hyphae more rapidly in the presence of serum, and had a propensity to grow as elongated cells or pseudohyphae in Ca²⁺-depleted medium, on SD, or on medium containing cell wall perturbing compounds. This suggests that depletion of Ca²⁺-uptake perturbs yeast-hypha morphogenesis, perhaps by inducing a nutrient starvation stress response. The <i>mid1</i> mutant and a number of other <i>C. albicans</i> Ca²⁺ signalling mutants were defective in chlamydospore formation, suggesting a role for Ca²⁺ in two morphogenetic genesises: the hyphae and of chlamydospores. The role of Mid1p in the thigmotropic reorientation responses of <i>C. albicans</i> hyphae was investigated. The <i>mid1</i> mutant strain displayed reduced ability to reorientate growth upon contact with ridges on an etched quartz slide. Suggesting that Mid1p may function as stretch-activated Ca²⁺ channel in <i>C. albicans</i>. The ability of <i>C. albicans</i> <i>mid1</i> mutants to respond to an electric field was also attenuated, suggesting that Mid1p may form part of a voltage-sensitive Ca²⁺ channel in <i>C. albicans</i> that plays a central role in the steering mechanism of <i>C. albicans</i> hyphae. The <i>C. albicans </i>kinase Cst20p may function downstream of Mid1p in growth reorientation responses.

Ca2+ signals determine smooth muscle contraction and the switch from a contractile to a migratory-proliferative phenotype(s), which requires changes in gene expression. However, the mechanism by which different Ca2+ signals are selective for these processes is enigmatic. In the thesis, I built on the “panjunctional sarcoplasmic reticulum” hypothesis, and described the evidence in support of the view that a variety of Ca2+ pumps and release channels, with different kinetics and affinities for Ca2+, are strategically positioned within the cytoplasmic nanocourses of pulmonary arterial smooth muscle cells (PASMCs), and they serve to demarcate different Ca2+ signalling. Nanocourses of the SR are formed in the perinuclear, extraperinuclear, subplasmalemmal regions and the nucleus. Different subtypes of ryanodine receptors (RyRs) are targeted to those nanocourses. Immunocytochemistry results suggest that RyR1s was preferentially targeted to the subplasmalemmal and nuclear nanocourses of PASMCs, they gave rise to a spatially restricted Ca2+ signal within the nanocourses upon stimulation, without affecting global Ca2+ concentration. The Ca2+ signals in the subplasmalemmal nanocourses were shown to induce arterial smooth muscle cell relaxation. On the other hand, the RyR2 and 3 were shown to target to the perinuclear and extraperinuclear nanocourses. Upon stimulation, they generate propagating Ca2+ waves in the cytoplasmic nanocourses, which trigger arterial smooth muscle cell contraction. However, during this process, no Ca2+ transient was observed within the subplasmalemmal nanocourses, suggesting that the regulation of both contraction and relaxation of smooth muscle cells are achieved by spatially restricted Ca2+ signals within different nanocourses. Invaginations of the nucleoplasmic reticulum in arterial myocytes form trans-nuclear networks of cytoplasmic nanospaces, generate Ca2+ signals by strategically positioned Ca2+ pumps (SERCA1) and release channels (RyR1). Within a subpopulation of nuclear invaginations, evoked Ca2+ signals via ryanodine receptors exhibited spatial and temporal separation from adjacent Ca2+ signals within a single “activated” nuclear invagination, and also from those Ca2+ signals arising within different nuclear invaginations. Moreover, nuclear invaginations provide sites for transcriptional suppression, because lamin A and/or emerin line the entire surface of their inner nuclear membranes and co-localise with nesprin-1 positive puncta. More intriguing still, a subpopulation of these nuclear invaginations harboured punctate regions of colocalisation between lamin A and the suppressive heterochromatin mark H3K9me2, while emerin-positive invaginations harboured puncta of BAF (Barrior to autointegration factor) co-localisation and thus an alternative pathway to the regulation of gene expression. I propose that nuclear invaginations form cytoplasmic nanotubes within which nano-patterning of Ca2+ signals may support stochastic modulation of transcriptional suppressors. Together, the cytoplasmic nanocourses form a cell-wide web for Ca2+ signalling and the regulation of various arterial smooth muscle functions, ranging from the regulation of blood pressure by vasodilation and vasoconstriction to gene expression.

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P2X4 receptors (P2X4R) are ligand-gated ion channels activated by ATP and with a high permeability to Ca2+. They are predominantly localised to lysosomes and from there can traffic to the cell surface. ATP levels within the lysosome are high but P2X4Rs are inhibited by the acidic pH. Previously, it was shown that the alkalinisation of lysosomes using pharmacological reagents was sufficient to activate P2X4Rs, which promoted homotypic lysosome fusion. The main aim of this study was to identify physiological regulators of lysosomal P2X4Rs and to examine their role in lysosome Ca2+ signalling and fusion. The first candidate I investigated was P2X7R, which is typically co-expressed with P2X4R in immune and epithelial cells, and which has already been shown to induce changes in lysosome properties upon activation. I co-expressed these two receptors in normal rat kidney (NRK) cells and in HeLa cells and looked for a synergistic interaction between them in promoting lysosome fusion, as assessed by measuring the size of lysosomes. My results showed a significant increase in lysosome size following activation of P2X7R but only in the presence of P2X4R. Neither receptor alone was sufficient to promote lysosome fusion in response to the agonist BzATP. LAMP-GECO was used to measure changes in cytosolic [Ca2+] within the vicinity of the lysosome. Fusion of the Ca2+ reporter (GECO) to the C-terminus of LAMP-1 targets GECO to the cytosolic surface of the lysosome. Co-expression of P2X4R with P2X7R augmented the P2X7R-induced Ca2+ signal suggesting that P2X4Rs mediate lysosomal Ca2+ efflux downstream of P2X7R stimulation. Next, I showed that the expression of P2X4R was sufficient to enhance the cytosolic Ca2+ response to the activation of endogenous histamine H1 receptors and to promote lysosome fusion. Similar results were obtained with P2Y2R stimulation, which also couples to the phospholipase C pathway. Further experiments were conducted to look at differences in the trafficking behaviour of human and rat P2X4Rs and to examine a role for P2X4Rs in autophagic flux. My results suggest a synergistic interaction between P2X4R and P2X7R which inhibits autophagic flux, similar to the effect of bafilomycin treatment. Therefore, the effect of P2X4/7R in autophagy may be mediated by the alkalinisation of lysosomes. Altogether the results of my project improve our understanding of how the P2X4R Ca2+ channel regulates lysosome function.

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Previous investigations on pulmonary artery smooth muscle cells have shown that nicotinic acid adenine dinucleotide diphosphate (NAADP) evokes highly localised intracellular Ca²⁺ bursts by mobilising thapsigargin-insensitive Ca²⁺ stores. Such localised Ca²⁺ signals may initiate global Ca²⁺ waves and contraction of the myocytes through the recruitment of ryanodine receptors (RyR) located on the sarcoplasmic reticulum (SR) via Ca²⁺-induced Ca²⁺-release (CICR). In this thesis I have shown that NAADP evokes localised Ca²⁺ signals through the mobilisation of a bafilomycin A1-sensitive, lysosome-related Ca²⁺ store. Lysosomal Ca²⁺ stores facilitate this process by colocalising with a subpopulation of RyRs on the surface of the SR to comprise a highly specialised trigger zone for Ca²⁺ signalling by NAADP. I have also shown that the proposed trigger zone for NAADP-dependent Ca²⁺ signalling may be formed between lysosomes and clusters of RyR subtype 3 (RyR3) located in close proximity to one another in the perinuclear region of cells. Localised Ca²⁺ bursts generated by NAADP-dependent Ca²⁺ release from acidic Ca²⁺ stores and subsequent CICR via RyR3 on the SR may then amplify Ca²⁺ bursts into a propagating Ca²⁺ signal by recruiting clusters of RyR subtype 2 (RyR2) located in the perinuclear and extra-perinuclear regions of the cell. The presence of this trigger zone may explain, in part, why Ca²⁺ bursts by NAADP induce, in an all-or-none manner, global Ca²⁺ signals by CICR via RyRs on the SR. Consistent with a role for NAADP and lysosomes as a discrete and agonist-specific Ca²⁺ signalling pathway utilised by vasoconstrictors, I have shown that endothelin-1 (ET-1), but not phenylephrine or prostaglandin-F2α, mobilises Ca²⁺ stores by NAADP, and that ET-1 initiates Ca²⁺ signalling by NAADP in a receptor subtype-specific manner through the activation of ETB receptors. These findings further advance our understanding of how that spatial organisation of discrete, organellar Ca²⁺ stores underpin the generation of differential Ca²⁺ signalling patterns by different Ca²⁺-mobilising messengers.