Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin

• Published in: Molecular microbiology Vol. 52; no. 5; pp. 1437 - 1450
• Main Authors: Nelson, G., Kozlova‐Zwinderman, O., Collis, A. J., Knight, M. R., Fincham, J. R. S., Stanger, C. P., Renwick, A., Hessing, J. G. M., Punt, P. J., Van Den Hondel, C. A. M. J. J., Read, N. D.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-06-2004; Blackwell Science; Blackwell Publishing Ltd
• Subjects: Aequorin - genetics; Aequorin - metabolism; Aspergillus - cytology; Aspergillus - genetics; Aspergillus - metabolism; Base Sequence; Biological and medical sciences; Calcimycin - metabolism; Calcium - metabolism; Calcium Channel Blockers - metabolism; Calcium Signaling - physiology; Chelating Agents - metabolism; Codon; Egtazic Acid - analogs & derivatives; Egtazic Acid - metabolism; Enzyme Inhibitors - metabolism; Fundamental and applied biological sciences. Psychology; Indoles - metabolism; Ionophores - metabolism; Microbiology; Miscellaneous; Molecular Sequence Data; Mycology; Neurospora crassa; Neurospora crassa - cytology; Neurospora crassa - genetics; Neurospora crassa - metabolism; Osmotic Pressure; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Stress, Mechanical; Fungi; Codon; Calcium; Microbiology; Enzyme; Oxidoreductases; Photoprotein; Renilla-luciferin 2-monooxygenase; Thallophyta
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/j.1365-2958.2004.04066.x

Summary
Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo‐osmotic shock and high external calcium) were found transiently to increase [Ca2+]c. Each of the calcium signatures associated with these physico‐chemical treatments was unique, suggesting the involvement of three distinct calcium‐mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca2+]c responses to hypo‐osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca2+]c responses to mechanical perturbation and hypo‐osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca2+]c levels.