| Summary: | The aim of this study was to monitor changes in cytosolic free calcium ([Ca2+]c in filamentous fungi using codon-optimised aequorin under different conditions In order to analyse Ca 2+ signalling in these organisms, and to use the recombinant aequorin method in fungicide mode-of-action studies, and as novel toxicant biosensor. Calcium signalling is little understood in filamentous fungi largely because easv and routine methods for Ca 2+ measurement in living hyphae have previously been unavailable. Recently a new method for measuring cellular Ca 2+ based on using codon-optimised recombinant aequorin, has been developed and used throughout the present study. The calibration method to convert light detected from aequorin expressing strains into [Ca 2+]c concentrations was optimised and critically evaluated. It was concluded that codon-optimised aequorin can provide excellent qualitative measurements of fungal [Ca2+]c but that precise quantification of [Ca2+]c using this method need to be treated with caution. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external CaC12, but not hyper-osmotic shock) were found to transiently increase [Ca 2+]c levels and to generate specific [Ca 2+]c signatures. Different parameters of the Ca 2+ signature (rise time, amplitude and full width half maximum) were quantified. Transient [Ca2+]c increases were also observed in response to cold and heat shock. Using Ca2+ channel blockers (LaC13, KP4, ryanodine, nifedipine, TN1B-8, verapamil), the Ca 2+ chelator BAPTA, and [Ca2+]c agonists (A23187, caffeine and cyclopiazonic acid), it was shown that the [Ca 2+]c increases resulting from hypo-osmotic shock and high external CaCl2 are predominantly due to the influx of Ca 2+ from the external media through plasma membrane Ca 2+ channels. The [Ca 2+ 1C increases resulting from mechanical perturbation seem to arise from both extracellular and intracellular sources. My results indicate that filamentous fungi possess a number of the components of the calcium signalling machinery found in other eukaryotic cells.
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