Ca²⁺/Calmodulin signalling during colony initiation in Neurospora crassa
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The primary research aims of this thesis were to analyse the mechanism of Ca²⁺/calmodulin (CaM) signalling during conidial germination and conidial anastomosis tube (CAT)-mediated fusion in Neurospora crassa. Ca²⁺ is an ubiquitous signalling molecule that regulates many important processes in filamentous fungi including spore germination, hyphal growth, mechanosensing, stress responses, circadian rhythms, and the virulence of pathogens. Transient increases in cytosolic free calcium ([Ca²⁺]c) act as intracellular signals. As the primary intracellular Ca²⁺ receptor, calmodulin (CaM) converts these Ca²⁺ signals into responses by regulating the activities of numerous target proteins. Ca²⁺-free medium, antagonists of L-type Ca²⁺ channels, CaM and calcineurin were found to inhibit CAT fusion. In addition, my results showed that CAT chemotropism is dependent on extracellular Ca²⁺. 65 genes were identified as likely components of the Ca²⁺ signalling machinery of N. crassa based on a comparative genomic analysis of S. cerevisiae, A. fumigatus and C. albicans. Deletion mutants of 29 of these genes were characterized in relation to their possible roles during colony initiation and development. Four of these mutants (Δcna-1, Δcnb-1, Δcamk-1, Δplc-2, and Δrgs-1), which were homokaryons, exhibited strong morphological phenotypes associated with CAT fusion. To identify the protein machinery involved in Ca²⁺/CaM signalling during colony initiation, proteins that directly or indirectly interacted with CaM were isolated from germlings by immunoprecipitation and analyzed by mass spectroscopy. A total of 286 putative Ca²⁺/CaM-interacting proteins were identified in this way and 30 of these proteins contained CaM-binding motifs. This proteomics analysis provided evidence for Ca²⁺/CaM signalling playing a role in regulating the activity of a wide range of proteins including MAP kinases in the cell integrity pathway, Ras/Rho signalling pathway, and microtubule and actin cytoskeletal proteins. GFP labelled CaM localized as dynamic spots associated with the plasma membrane and cytoplasm in both germ tubes and CATs. Significant CaM accumulation was observed in the tips of CATs growing towards each other, around fusion pores at sites of CAT fusion, and at developing septa in germ tubes. CaM localization was influenced by the actin and microtubule cytoskeleton during the colony initiation. Inhibition of F-actin polymerization with latrunculin-A suppressed the pronounced accumulation of CaM at growing germ tube and CAT tips. The movement of CaM associated with spindle pole bodies was prevented by treatment with the microtubule polymerization inhibitor benomyl. The absence of myo-5 resulted in reduced CAT fusion and the lack recruitment of CaM at growing tips indicating a role for the motor protein, myosin-5, in these processes. Finally, by expressing the genetically encoded Ca²⁺ sensor GCaMP6s under the control of tef-1 promoter in N. crassa, I have been able to image [Ca²⁺]c dynamics in this fungus for the first time. Using this I have been able to detect localized [Ca²⁺]c spikes and waves in conidia, germ tubes and CATs. However, I obtained no clear evidence for localized [Ca²⁺]c changes being associated with CAT chemotropism or fusion.