Title
Calcium measurements in living filamentous fungi expressing codon-optimized aequorin
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.
van den Hondel, C.A.M.J.J.
Read, N.D.
TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Publication year
2004
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.
Subject
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
To reference this document use:
http://resolver.tudelft.nl/uuid:980c6c6d-ca17-4e4b-89bf-55c7b46b009c
DOI
https://doi.org/10.1111/j.1365-2958.2004.04066.x
TNO identifier
237829
ISSN
0950-382X
Source
Molecular Microbiology, 52 (5), 1437-1450
Document type
article