Calcium measurements in living filamentous fungi expressing codon-optimised aequorin

Abstract

The aim of this study was to monitor changes in cytosolic free calcium ([Ca²⁺]꜀ in filamentous fungi using codon-optimised aequorin under different conditions in order to analyse Ca²⁺ 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 Ca²⁺ measurement in living hyphae have previously been unavailable. Recently a new method for measuring cellular Ca²⁺ 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 [Ca²⁺]꜀ concentrations was optimised and critically evaluated. It was concluded that codon-optimised aequorin can provide excellent qualitative measurements of fungal [Ca²⁺]꜀ using this method need to be treated with caution.

Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external CaCl₂, but not hyper-osmotic shock) were found to transiently increase [Ca²⁺]꜀ levels and to generate specific [Ca²⁺]꜀ signatures. Different parameters of the Ca²⁺ signature (rise time, amplitude and full width half maximum) were quantified. Transient [Ca²⁺]꜀ increases were also observed in response to cold and heat shock. Using Ca²⁺ channel blockers (LaCl₃, KP4, ryanodine, nifedipine, TMB-8, verapamil), the Ca²⁺ chelator BAPTA, and Ca²⁺ agonists (A23187, caffeine and cyclopiazonic acid), it was shown that the [Ca²⁺]꜀ increases resulting from hypo-osmotic shock and high external CaCl₂, are predominantly due to the influx of Ca²⁺ from the external media through plasma membrane Ca²⁺ channels. The [Ca²⁺]꜀ 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.

Differences were noted in the [Ca²⁺]꜀ responses to physico-chemicals stimuli and Ca²⁺ agonists in fungi grown in liquid medium compared with those grown on solid medium.

[Ca²⁺]꜀ plays an important role in signal-response coupling. I therefore investigated whether the external stimuli shown to transiently elevate [Ca²⁺]꜀ also induced changes in hyphal tip morphology, branching frequency, colony extension rate and sporulation. The short term increases in [Ca²⁺]꜀ resulting from mechanical perturbation, hypo-osmotic shock and high external CaCl₂ were found not to influence any of theses parameters. However the long term increase in [Ca²⁺]꜀ caused by A23187 resulted in the formation of bulbous hyphal compartments and hyperbranching. A23187 also inhibited growth of A.awamori and caused significant cell death.

The aequorin method was found to be useful in fungicide mode-of-action studies. A large number of commercial fungicides were shown to perturb [Ca²⁺]꜀ homeostasis. Evidence was obtained which suggested that a cell permeabilizing compound (viscosinamide) produced by antagonistic soil bacteria may inhibit the growth of soil fungi by perturbing Ca²⁺ signalling or [Ca²⁺]꜀ homeostasis.

The aequorin method was used as a novel eukaryotic toxicant biosensor. Three environmental pollutants (3,5-DCP, Cr³⁺ and Zn²⁺) were tested and were each shown to influence in a unique manner the Ca²⁺ -signature in response to the addition of external CaCl₂. Preliminary results suggested that the fungal aequorin biosensor may be less sensitive for detecting these pollutants than the standard Vibrio fischeri luciferase biosensor.