Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin
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 obtaine...
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| Veröffentlicht in: | Molecular microbiology 2004-06, Vol.52 (5), p.1437-1450 |
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| creator | 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. |
| description | 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. |
| doi_str_mv | 10.1111/j.1365-2958.2004.04066.x |
| format | Article |
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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.</description><identifier>ISSN: 0950-382X</identifier><identifier>EISSN: 1365-2958</identifier><identifier>DOI: 10.1111/j.1365-2958.2004.04066.x</identifier><identifier>PMID: 15165245</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Science Ltd</publisher><subject>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</subject><ispartof>Molecular microbiology, 2004-06, Vol.52 (5), p.1437-1450</ispartof><rights>2004 INIST-CNRS</rights><rights>Copyright Blackwell Scientific Publications Ltd. Jun 2004</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5016-f240c11136ab1da52cdcf1cab90b12b3d8f5981f2433fcdba5a05ebf79a7b6bb3</citedby><cites>FETCH-LOGICAL-c5016-f240c11136ab1da52cdcf1cab90b12b3d8f5981f2433fcdba5a05ebf79a7b6bb3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1365-2958.2004.04066.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1365-2958.2004.04066.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,1427,27901,27902,45550,45551,46384,46808</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=15792941$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15165245$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nelson, G.</creatorcontrib><creatorcontrib>Kozlova‐Zwinderman, O.</creatorcontrib><creatorcontrib>Collis, A. J.</creatorcontrib><creatorcontrib>Knight, M. R.</creatorcontrib><creatorcontrib>Fincham, J. R. S.</creatorcontrib><creatorcontrib>Stanger, C. P.</creatorcontrib><creatorcontrib>Renwick, A.</creatorcontrib><creatorcontrib>Hessing, J. G. M.</creatorcontrib><creatorcontrib>Punt, P. J.</creatorcontrib><creatorcontrib>Van Den Hondel, C. A. M. J. J.</creatorcontrib><creatorcontrib>Read, N. D.</creatorcontrib><title>Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin</title><title>Molecular microbiology</title><addtitle>Mol Microbiol</addtitle><description>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.</description><subject>Aequorin - genetics</subject><subject>Aequorin - metabolism</subject><subject>Aspergillus - cytology</subject><subject>Aspergillus - genetics</subject><subject>Aspergillus - metabolism</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Calcimycin - metabolism</subject><subject>Calcium - metabolism</subject><subject>Calcium Channel Blockers - metabolism</subject><subject>Calcium Signaling - physiology</subject><subject>Chelating Agents - metabolism</subject><subject>Codon</subject><subject>Egtazic Acid - analogs & derivatives</subject><subject>Egtazic Acid - metabolism</subject><subject>Enzyme Inhibitors - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Indoles - metabolism</subject><subject>Ionophores - metabolism</subject><subject>Microbiology</subject><subject>Miscellaneous</subject><subject>Molecular Sequence Data</subject><subject>Mycology</subject><subject>Neurospora crassa</subject><subject>Neurospora crassa - cytology</subject><subject>Neurospora crassa - genetics</subject><subject>Neurospora crassa - metabolism</subject><subject>Osmotic Pressure</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Alignment</subject><subject>Stress, Mechanical</subject><issn>0950-382X</issn><issn>1365-2958</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1O3DAURq0KxEyBV6iiSnSX4J_YiRcsqlEpI4HYgMTOsh175FHiDPaEznTFI_QZ-yQ4nVGp2BRvbPme--leHQAyBAuUzvmyQITRHHNaFxjCsoAlZKzYfADTv4UDMIWcwpzU-GECPsa4hBARyMgRmCCKGMUlnYL7mWy1G7qsMzIOwXTGrzPns9Y9Ob_IrGvl-NUPMbODX7jMbFbBxDgWdd_0_vfzr361dp37aZpMmsehD86fgEMr22hO9_cxuL_8dje7yq9vv89nX69zTSFiucUl1GkfwqRCjaRYN9oiLRWHCmFFmtpSXqOEEWJ1oySVkBplKy4rxZQix-DLLncV-sfBxLXoXNSmbaU3aWRRIc5IVeL_gqjivCxZlcDPb8BlPwSflhApiyJIGU1QvYN06GMMxopVcJ0MW4GgGAWJpRg9iNGDGAWJP4LEJrV-2ucPqjPNa-PeSALO9oCMWrY2SK9d_IerOOYlStzFjvvhWrN99wDi5mY-vsgLC12uNg</recordid><startdate>200406</startdate><enddate>200406</enddate><creator>Nelson, G.</creator><creator>Kozlova‐Zwinderman, O.</creator><creator>Collis, A. J.</creator><creator>Knight, M. R.</creator><creator>Fincham, J. R. S.</creator><creator>Stanger, C. P.</creator><creator>Renwick, A.</creator><creator>Hessing, J. G. M.</creator><creator>Punt, P. J.</creator><creator>Van Den Hondel, C. A. M. J. J.</creator><creator>Read, N. D.</creator><general>Blackwell Science Ltd</general><general>Blackwell Science</general><general>Blackwell Publishing Ltd</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>200406</creationdate><title>Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin</title><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.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5016-f240c11136ab1da52cdcf1cab90b12b3d8f5981f2433fcdba5a05ebf79a7b6bb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Aequorin - genetics</topic><topic>Aequorin - metabolism</topic><topic>Aspergillus - cytology</topic><topic>Aspergillus - genetics</topic><topic>Aspergillus - metabolism</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Calcimycin - metabolism</topic><topic>Calcium - metabolism</topic><topic>Calcium Channel Blockers - metabolism</topic><topic>Calcium Signaling - physiology</topic><topic>Chelating Agents - metabolism</topic><topic>Codon</topic><topic>Egtazic Acid - analogs & derivatives</topic><topic>Egtazic Acid - metabolism</topic><topic>Enzyme Inhibitors - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Indoles - metabolism</topic><topic>Ionophores - metabolism</topic><topic>Microbiology</topic><topic>Miscellaneous</topic><topic>Molecular Sequence Data</topic><topic>Mycology</topic><topic>Neurospora crassa</topic><topic>Neurospora crassa - cytology</topic><topic>Neurospora crassa - genetics</topic><topic>Neurospora crassa - metabolism</topic><topic>Osmotic Pressure</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Alignment</topic><topic>Stress, Mechanical</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nelson, G.</creatorcontrib><creatorcontrib>Kozlova‐Zwinderman, O.</creatorcontrib><creatorcontrib>Collis, A. J.</creatorcontrib><creatorcontrib>Knight, M. R.</creatorcontrib><creatorcontrib>Fincham, J. R. S.</creatorcontrib><creatorcontrib>Stanger, C. P.</creatorcontrib><creatorcontrib>Renwick, A.</creatorcontrib><creatorcontrib>Hessing, J. G. M.</creatorcontrib><creatorcontrib>Punt, P. J.</creatorcontrib><creatorcontrib>Van Den Hondel, C. A. M. J. J.</creatorcontrib><creatorcontrib>Read, N. D.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Molecular microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nelson, G.</au><au>Kozlova‐Zwinderman, O.</au><au>Collis, A. J.</au><au>Knight, M. R.</au><au>Fincham, J. R. S.</au><au>Stanger, C. P.</au><au>Renwick, A.</au><au>Hessing, J. G. M.</au><au>Punt, P. J.</au><au>Van Den Hondel, C. A. M. J. J.</au><au>Read, N. D.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin</atitle><jtitle>Molecular microbiology</jtitle><addtitle>Mol Microbiol</addtitle><date>2004-06</date><risdate>2004</risdate><volume>52</volume><issue>5</issue><spage>1437</spage><epage>1450</epage><pages>1437-1450</pages><issn>0950-382X</issn><eissn>1365-2958</eissn><abstract>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.</abstract><cop>Oxford, UK</cop><pub>Blackwell Science Ltd</pub><pmid>15165245</pmid><doi>10.1111/j.1365-2958.2004.04066.x</doi><tpages>14</tpages><oa>free_for_read</oa></addata></record> |
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| 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 |
| title | Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin |
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