Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins
Pollard and colleagues demonstrate in vitro and in fission yeast that the LifeAct actin probe can affect actin filament nucleation and dynamics and perturb actin-dependent cellular processes unless low concentrations are used. When tagged with a fluorescent protein, actin is not fully functional 1 ,...
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| Veröffentlicht in: | Nature cell biology 2016-06, Vol.18 (6), p.676-683 |
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| creator | Courtemanche, Naomi Pollard, Thomas D. Chen, Qian |
| description | Pollard and colleagues demonstrate
in vitro
and in fission yeast that the LifeAct actin probe can affect actin filament nucleation and dynamics and perturb actin-dependent cellular processes unless low concentrations are used.
When tagged with a fluorescent protein, actin is not fully functional
1
, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells
2
. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly
in vitro
and in fission yeast cells. mEGFP–LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP–LifeAct and LifeAct–mCherry bind actin filaments with
K
d
values of ∼10 μM. LifeAct–mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct–mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts. |
| doi_str_mv | 10.1038/ncb3351 |
| format | Article |
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in vitro
and in fission yeast that the LifeAct actin probe can affect actin filament nucleation and dynamics and perturb actin-dependent cellular processes unless low concentrations are used.
When tagged with a fluorescent protein, actin is not fully functional
1
, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells
2
. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly
in vitro
and in fission yeast cells. mEGFP–LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP–LifeAct and LifeAct–mCherry bind actin filaments with
K
d
values of ∼10 μM. LifeAct–mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct–mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.</description><identifier>ISSN: 1465-7392</identifier><identifier>EISSN: 1476-4679</identifier><identifier>DOI: 10.1038/ncb3351</identifier><identifier>PMID: 27159499</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>14 ; 14/19 ; 14/63 ; 631/1647/245 ; 631/80/128/1276 ; 631/80/313/1461 ; 631/80/641/2090 ; 82 ; 82/83 ; Actin ; Actin Cytoskeleton - metabolism ; Actin Depolymerizing Factors - metabolism ; Actins - metabolism ; Animals ; Biology ; Cancer Research ; Cell Biology ; Cell culture ; Cell Cycle Proteins - metabolism ; Cytokinesis - physiology ; Cytoskeletal Proteins - metabolism ; Developmental Biology ; Endocytosis - physiology ; Innovations ; letter ; Life Sciences ; Peptides ; Profilins - metabolism ; Properties ; Proteins ; Schizosaccharomyces - metabolism ; Schizosaccharomyces pombe Proteins - metabolism ; Stem Cells ; Vitamin B ; Yeast ; Yeasts</subject><ispartof>Nature cell biology, 2016-06, Vol.18 (6), p.676-683</ispartof><rights>Springer Nature Limited 2016</rights><rights>COPYRIGHT 2016 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Jun 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c476t-fd787e26514c00ee450d3e77dfb6aff8ea61754356114c3093b9e6aa6f2f534e3</citedby><cites>FETCH-LOGICAL-c476t-fd787e26514c00ee450d3e77dfb6aff8ea61754356114c3093b9e6aa6f2f534e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/ncb3351$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/ncb3351$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27159499$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Courtemanche, Naomi</creatorcontrib><creatorcontrib>Pollard, Thomas D.</creatorcontrib><creatorcontrib>Chen, Qian</creatorcontrib><title>Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins</title><title>Nature cell biology</title><addtitle>Nat Cell Biol</addtitle><addtitle>Nat Cell Biol</addtitle><description>Pollard and colleagues demonstrate
in vitro
and in fission yeast that the LifeAct actin probe can affect actin filament nucleation and dynamics and perturb actin-dependent cellular processes unless low concentrations are used.
When tagged with a fluorescent protein, actin is not fully functional
1
, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells
2
. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly
in vitro
and in fission yeast cells. mEGFP–LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP–LifeAct and LifeAct–mCherry bind actin filaments with
K
d
values of ∼10 μM. LifeAct–mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct–mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.</description><subject>14</subject><subject>14/19</subject><subject>14/63</subject><subject>631/1647/245</subject><subject>631/80/128/1276</subject><subject>631/80/313/1461</subject><subject>631/80/641/2090</subject><subject>82</subject><subject>82/83</subject><subject>Actin</subject><subject>Actin Cytoskeleton - metabolism</subject><subject>Actin Depolymerizing Factors - metabolism</subject><subject>Actins - metabolism</subject><subject>Animals</subject><subject>Biology</subject><subject>Cancer Research</subject><subject>Cell Biology</subject><subject>Cell culture</subject><subject>Cell Cycle Proteins - metabolism</subject><subject>Cytokinesis - physiology</subject><subject>Cytoskeletal Proteins - metabolism</subject><subject>Developmental Biology</subject><subject>Endocytosis - physiology</subject><subject>Innovations</subject><subject>letter</subject><subject>Life Sciences</subject><subject>Peptides</subject><subject>Profilins - metabolism</subject><subject>Properties</subject><subject>Proteins</subject><subject>Schizosaccharomyces - metabolism</subject><subject>Schizosaccharomyces pombe Proteins - metabolism</subject><subject>Stem Cells</subject><subject>Vitamin B</subject><subject>Yeast</subject><subject>Yeasts</subject><issn>1465-7392</issn><issn>1476-4679</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNptkm1vFCEQxzfGxtZq_AaGxBfVF1theTpeXpqqTS4x8eE14djhSt2FE9hq--ll07P1GgMBMvObPzMwTfOK4FOC6eJ9sGtKOXnSHBEmRcuEVE_ns-CtpKo7bJ7nfIUxYQzLZ81hJwlXTKmj5sfyOvrehw0yqYAztmT06xICsnEKZbZv43AzQvK30KPq9gHVOfhrQBaGodK-XKKVd7C0BbkpV6xE5IYpJsgWQkHbFAv4kF80B84MGV7u9uPm-4fzb2ef2tXnjxdny1Vra-6ldb1cSOgEJ8xiDMA47ilI2bu1MM4twAgiOaNckEpQrOhagTBGuM5xyoAeN2_vdOvFPyfIRY8-z8maAHHKmkjVUVlXWtE3j9CrOKVQs5sporBQnXqgNmYA7YOLJRk7i-ol4wsuKzhTp_-h6uhh9DYGcL7a9wLe7QVUpsDvsjFTzvri65d99uSOtSnmnMDpbfKjSTeaYD23gN61QCVf70qa1iP099zfP394nlxdYQPpn5ofaf0BN-S3lg</recordid><startdate>20160601</startdate><enddate>20160601</enddate><creator>Courtemanche, Naomi</creator><creator>Pollard, Thomas D.</creator><creator>Chen, Qian</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><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>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20160601</creationdate><title>Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins</title><author>Courtemanche, Naomi ; Pollard, Thomas D. ; Chen, Qian</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c476t-fd787e26514c00ee450d3e77dfb6aff8ea61754356114c3093b9e6aa6f2f534e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>14</topic><topic>14/19</topic><topic>14/63</topic><topic>631/1647/245</topic><topic>631/80/128/1276</topic><topic>631/80/313/1461</topic><topic>631/80/641/2090</topic><topic>82</topic><topic>82/83</topic><topic>Actin</topic><topic>Actin Cytoskeleton - metabolism</topic><topic>Actin Depolymerizing Factors - metabolism</topic><topic>Actins - metabolism</topic><topic>Animals</topic><topic>Biology</topic><topic>Cancer Research</topic><topic>Cell Biology</topic><topic>Cell culture</topic><topic>Cell Cycle Proteins - metabolism</topic><topic>Cytokinesis - physiology</topic><topic>Cytoskeletal Proteins - metabolism</topic><topic>Developmental Biology</topic><topic>Endocytosis - physiology</topic><topic>Innovations</topic><topic>letter</topic><topic>Life Sciences</topic><topic>Peptides</topic><topic>Profilins - metabolism</topic><topic>Properties</topic><topic>Proteins</topic><topic>Schizosaccharomyces - metabolism</topic><topic>Schizosaccharomyces pombe Proteins - metabolism</topic><topic>Stem Cells</topic><topic>Vitamin B</topic><topic>Yeast</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Courtemanche, Naomi</creatorcontrib><creatorcontrib>Pollard, Thomas D.</creatorcontrib><creatorcontrib>Chen, Qian</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Nature cell biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Courtemanche, Naomi</au><au>Pollard, Thomas D.</au><au>Chen, Qian</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins</atitle><jtitle>Nature cell biology</jtitle><stitle>Nat Cell Biol</stitle><addtitle>Nat Cell Biol</addtitle><date>2016-06-01</date><risdate>2016</risdate><volume>18</volume><issue>6</issue><spage>676</spage><epage>683</epage><pages>676-683</pages><issn>1465-7392</issn><eissn>1476-4679</eissn><abstract>Pollard and colleagues demonstrate
in vitro
and in fission yeast that the LifeAct actin probe can affect actin filament nucleation and dynamics and perturb actin-dependent cellular processes unless low concentrations are used.
When tagged with a fluorescent protein, actin is not fully functional
1
, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells
2
. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly
in vitro
and in fission yeast cells. mEGFP–LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP–LifeAct and LifeAct–mCherry bind actin filaments with
K
d
values of ∼10 μM. LifeAct–mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct–mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>27159499</pmid><doi>10.1038/ncb3351</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 14 14/19 14/63 631/1647/245 631/80/128/1276 631/80/313/1461 631/80/641/2090 82 82/83 Actin Actin Cytoskeleton - metabolism Actin Depolymerizing Factors - metabolism Actins - metabolism Animals Biology Cancer Research Cell Biology Cell culture Cell Cycle Proteins - metabolism Cytokinesis - physiology Cytoskeletal Proteins - metabolism Developmental Biology Endocytosis - physiology Innovations letter Life Sciences Peptides Profilins - metabolism Properties Proteins Schizosaccharomyces - metabolism Schizosaccharomyces pombe Proteins - metabolism Stem Cells Vitamin B Yeast Yeasts |
| title | Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins |
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