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Calcium measurements in living filamentous fungi expressing codon-optimized aequorin

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Author: 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. · Hondel, C.A.M.J.J. van den · Read, N.D.
Type:article
Date:2004
Institution: TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Source:Molecular Microbiology, 5, 52, 1437-1450
Identifier: 237829
doi: doi:10.1111/j.1365-2958.2004.04066.x
Keywords: Biology · Biotechnology · Aequorin · Calcimycin · Calcium · Calcium channel · Calcium channel blocking agent · Calcium channel stimulating agent · Cation · Chelating agent · Cyclopiazonic acid · Ethylene glycol 1,2 bis(2 aminophenyl) ether n,n,n',n' tetraacetic acid · Recombinant protein · 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid · Drug derivative · Egtazic acid · Enzyme inhibitor · Indole derivative · Ionophore · Aspergillus awamori · Aspergillus niger · Codon · Concentration (parameters) · Controlled study · Fungus · Measurement · Neurospora crassa · Nonhuman · Osmotic stress · Physical chemistry · Priority journal · Protein expression · Signal transduction · Stimulus · Stimulus response · Aspergillus · Calcium signaling · Cytology · Genetics · Mechanical stress · Metabolism · Molecular genetics · Nucleotide sequence · Osmotic pressure · Physiology · Sequence alignment · Aspergillus · Aspergillus awamori · Aspergillus niger · Fungi · Neurospora · Neurospora crassa · Aequorin · Aspergillus · Base Sequence · Calcimycin · Calcium · Calcium Channel Blockers · Calcium Signaling · Chelating Agents · Codon · Egtazic Acid · Enzyme Inhibitors · Indoles · Ionophores · Molecular Sequence Data · Neurospora crassa · Osmotic Pressure · Recombinant Proteins · Sequence Alignment · Stress, Mechanical

Abstract

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, hypoosmotic shock and high external calcium) were found transiently to increase [Ca 2+]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 [Ca 2+]c responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca 2+]c levels.