Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag
Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarit...
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| Veröffentlicht in: | Journal of Bacteriology 2012-12, Vol.194 (23), p.6382-6386 |
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| creator | Swulius, Matthew T Jensen, Grant J |
| description | Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFPSW) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, “patchy” localization patterns of MreB-RFPSW, even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered. |
| doi_str_mv | 10.1128/JB.00505-12 |
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The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFPSW) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, “patchy” localization patterns of MreB-RFPSW, even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.</description><identifier>ISSN: 0021-9193</identifier><identifier>EISSN: 1098-5530</identifier><identifier>EISSN: 1067-8832</identifier><identifier>DOI: 10.1128/JB.00505-12</identifier><identifier>PMID: 22904287</identifier><identifier>CODEN: JOBAAY</identifier><language>eng</language><publisher>Washington, DC: American Society for Microbiology</publisher><subject>actin ; bacteria ; Bacterial proteins ; Bacterial Proteins - analysis ; Bacteriological Techniques - methods ; Bacteriology ; Biological and medical sciences ; Cells ; chromosome segregation ; Chromosomes ; Cytoplasm ; cytoskeleton ; E coli ; Electron Microscope Tomography - methods ; Escherichia coli ; Escherichia coli - chemistry ; Escherichia coli Proteins - analysis ; Fluorescence ; fluorescence microscopy ; Fundamental and applied biological sciences. Psychology ; light microscopy ; Luminescent Proteins - analysis ; Microbiology ; Microscopy, Fluorescence - methods ; Miscellaneous ; Recombinant Fusion Proteins - analysis ; Red Fluorescent Protein ; Staining and Labeling - methods ; yellow fluorescent protein</subject><ispartof>Journal of Bacteriology, 2012-12, Vol.194 (23), p.6382-6386</ispartof><rights>2015 INIST-CNRS</rights><rights>Copyright American Society for Microbiology Dec 2012</rights><rights>Copyright © 2012, American Society for Microbiology. All Rights Reserved. 2012 American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c631t-aacd349b625aa963d6cfb344f75d60931997579923155149be6ce5e4dd8cace73</citedby><cites>FETCH-LOGICAL-c631t-aacd349b625aa963d6cfb344f75d60931997579923155149be6ce5e4dd8cace73</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497537/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497537/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27915,27916,53782,53784</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=26605716$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22904287$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Swulius, Matthew T</creatorcontrib><creatorcontrib>Jensen, Grant J</creatorcontrib><title>Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag</title><title>Journal of Bacteriology</title><addtitle>J Bacteriol</addtitle><description>Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFPSW) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, “patchy” localization patterns of MreB-RFPSW, even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.</description><subject>actin</subject><subject>bacteria</subject><subject>Bacterial proteins</subject><subject>Bacterial Proteins - analysis</subject><subject>Bacteriological Techniques - methods</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Cells</subject><subject>chromosome segregation</subject><subject>Chromosomes</subject><subject>Cytoplasm</subject><subject>cytoskeleton</subject><subject>E coli</subject><subject>Electron Microscope Tomography - methods</subject><subject>Escherichia coli</subject><subject>Escherichia coli - chemistry</subject><subject>Escherichia coli Proteins - analysis</subject><subject>Fluorescence</subject><subject>fluorescence microscopy</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>light microscopy</subject><subject>Luminescent Proteins - analysis</subject><subject>Microbiology</subject><subject>Microscopy, Fluorescence - methods</subject><subject>Miscellaneous</subject><subject>Recombinant Fusion Proteins - analysis</subject><subject>Red Fluorescent Protein</subject><subject>Staining and Labeling - methods</subject><subject>yellow fluorescent protein</subject><issn>0021-9193</issn><issn>1098-5530</issn><issn>1067-8832</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0s1v0zAYBvAIgVgZnLiDBUJCQtn82rEdX5DWqmObOkCiO3CyXMdp3LlxsVOm_vcktIyPCycf_NPj136cZc8BnwCQ8vRqfIIxwywH8iAbAZZlzhjFD7MRxgRyCZIeZU9SWmEMRcHI4-yIEIkLUopRtruw3hnt0XW0YzTZdSHdWm-70CLXomkyjY3ONE4jE7xD1xPAGJ9uZlOBLhPSLTqLnau16VCoUddY9DGf27h2bR_51Xof7tC534Zok7Fthz7H0Nk-eK6XT7NHtfbJPjusx9nN-XQ-uchnnz5cTs5mueEUulxrU9FCLjhhWktOK27qBS2KWrCKY0lBSsGElIQCY9BDy41ltqiq0mhjBT3O3u9zN9vF2lbDGFF7tYlureNOBe3U3zuta9QyfFf9qYLRIeDtISCGb1ubOrV2_W28160N26SAi5JRygT_PwUGoi-rHOjrf-gqbGP_bHtFheAF6dW7vTIxpBRtfT83YDW0r67G6mf7Cgb94s-r3ttfdffgzQHo1JdeR90al347zjETMAz3au8at2zuXLRKp7VaLRTIQhGqOC2H017uUa2D0svYB918IRjY8M9kQYH-AFEhyU8</recordid><startdate>20121201</startdate><enddate>20121201</enddate><creator>Swulius, Matthew T</creator><creator>Jensen, Grant J</creator><general>American Society for Microbiology</general><scope>FBQ</scope><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>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><scope>7S9</scope><scope>L.6</scope><scope>5PM</scope></search><sort><creationdate>20121201</creationdate><title>Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag</title><author>Swulius, Matthew T ; Jensen, Grant J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c631t-aacd349b625aa963d6cfb344f75d60931997579923155149be6ce5e4dd8cace73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>actin</topic><topic>bacteria</topic><topic>Bacterial proteins</topic><topic>Bacterial Proteins - analysis</topic><topic>Bacteriological Techniques - methods</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Cells</topic><topic>chromosome segregation</topic><topic>Chromosomes</topic><topic>Cytoplasm</topic><topic>cytoskeleton</topic><topic>E coli</topic><topic>Electron Microscope Tomography - methods</topic><topic>Escherichia coli</topic><topic>Escherichia coli - chemistry</topic><topic>Escherichia coli Proteins - analysis</topic><topic>Fluorescence</topic><topic>fluorescence microscopy</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>light microscopy</topic><topic>Luminescent Proteins - analysis</topic><topic>Microbiology</topic><topic>Microscopy, Fluorescence - methods</topic><topic>Miscellaneous</topic><topic>Recombinant Fusion Proteins - analysis</topic><topic>Red Fluorescent Protein</topic><topic>Staining and Labeling - methods</topic><topic>yellow fluorescent protein</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Swulius, Matthew T</creatorcontrib><creatorcontrib>Jensen, Grant J</creatorcontrib><collection>AGRIS</collection><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>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><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of Bacteriology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Swulius, Matthew T</au><au>Jensen, Grant J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag</atitle><jtitle>Journal of Bacteriology</jtitle><addtitle>J Bacteriol</addtitle><date>2012-12-01</date><risdate>2012</risdate><volume>194</volume><issue>23</issue><spage>6382</spage><epage>6386</epage><pages>6382-6386</pages><issn>0021-9193</issn><eissn>1098-5530</eissn><eissn>1067-8832</eissn><coden>JOBAAY</coden><abstract>Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFPSW) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, “patchy” localization patterns of MreB-RFPSW, even by standard light microscopy. 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| subjects | actin bacteria Bacterial proteins Bacterial Proteins - analysis Bacteriological Techniques - methods Bacteriology Biological and medical sciences Cells chromosome segregation Chromosomes Cytoplasm cytoskeleton E coli Electron Microscope Tomography - methods Escherichia coli Escherichia coli - chemistry Escherichia coli Proteins - analysis Fluorescence fluorescence microscopy Fundamental and applied biological sciences. Psychology light microscopy Luminescent Proteins - analysis Microbiology Microscopy, Fluorescence - methods Miscellaneous Recombinant Fusion Proteins - analysis Red Fluorescent Protein Staining and Labeling - methods yellow fluorescent protein |
| title | Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag |
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