Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos
Experimental and analytical methods are described for in vivo single-molecule imaging of GFP-tagged proteins at the cell surface and are applied to the developing C. elegans embryo. We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that ca...
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| Veröffentlicht in: | Nature methods 2014-06, Vol.11 (6), p.677-682 |
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| creator | Robin, François B McFadden, William M Yao, Baixue Munro, Edwin M |
| description | Experimental and analytical methods are described for
in vivo
single-molecule imaging of GFP-tagged proteins at the cell surface and are applied to the developing
C. elegans
embryo.
We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in
Caenorhabditis elegans
embryos. We exploited tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast, continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single-particle tracking (SPT). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface. |
| doi_str_mv | 10.1038/nmeth.2928 |
| format | Article |
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in vivo
single-molecule imaging of GFP-tagged proteins at the cell surface and are applied to the developing
C. elegans
embryo.
We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in
Caenorhabditis elegans
embryos. We exploited tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast, continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single-particle tracking (SPT). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.</description><identifier>ISSN: 1548-7091</identifier><identifier>EISSN: 1548-7105</identifier><identifier>DOI: 10.1038/nmeth.2928</identifier><identifier>PMID: 24727651</identifier><language>eng</language><publisher>New York: Nature Publishing Group US</publisher><subject>14/35 ; 631/136/1660 ; 631/57/2265 ; 631/80/2373 ; 631/80/85 ; 64/11 ; Analysis ; Animals ; Bioinformatics ; Biological Microscopy ; Biological Techniques ; Biomedical Engineering/Biotechnology ; Caenorhabditis elegans - embryology ; Caenorhabditis elegans - metabolism ; Caenorhabditis elegans Proteins - metabolism ; Cell physiology ; Cellular biology ; Dynamics ; Embryo, Nonmammalian ; Embryonic development ; Embryos ; Genetic aspects ; Green Fluorescent Proteins - metabolism ; Kinetics ; Life Sciences ; Membrane proteins ; Mobility ; Molecular Imaging ; Nematodes ; Photobleaching ; Physiological aspects ; Proteins ; Proteomics ; Surface Properties</subject><ispartof>Nature methods, 2014-06, Vol.11 (6), p.677-682</ispartof><rights>Springer Nature America, Inc. 2014</rights><rights>COPYRIGHT 2014 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Jun 2014</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c554t-5f3b6119c1f67397cf6f41886cca7cd179376ac2de52a51f67a334326feadbb83</citedby><cites>FETCH-LOGICAL-c554t-5f3b6119c1f67397cf6f41886cca7cd179376ac2de52a51f67a334326feadbb83</cites><orcidid>0000-0002-2420-2593</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/nmeth.2928$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/nmeth.2928$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,780,784,885,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24727651$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-03988994$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Robin, François B</creatorcontrib><creatorcontrib>McFadden, William M</creatorcontrib><creatorcontrib>Yao, Baixue</creatorcontrib><creatorcontrib>Munro, Edwin M</creatorcontrib><title>Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos</title><title>Nature methods</title><addtitle>Nat Methods</addtitle><addtitle>Nat Methods</addtitle><description>Experimental and analytical methods are described for
in vivo
single-molecule imaging of GFP-tagged proteins at the cell surface and are applied to the developing
C. elegans
embryo.
We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in
Caenorhabditis elegans
embryos. We exploited tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast, continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single-particle tracking (SPT). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. 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Academic</collection><collection>Hyper Article en Ligne (HAL)</collection><jtitle>Nature methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Robin, François B</au><au>McFadden, William M</au><au>Yao, Baixue</au><au>Munro, Edwin M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos</atitle><jtitle>Nature methods</jtitle><stitle>Nat Methods</stitle><addtitle>Nat Methods</addtitle><date>2014-06-01</date><risdate>2014</risdate><volume>11</volume><issue>6</issue><spage>677</spage><epage>682</epage><pages>677-682</pages><issn>1548-7091</issn><eissn>1548-7105</eissn><abstract>Experimental and analytical methods are described for
in vivo
single-molecule imaging of GFP-tagged proteins at the cell surface and are applied to the developing
C. elegans
embryo.
We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in
Caenorhabditis elegans
embryos. We exploited tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast, continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single-particle tracking (SPT). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.</abstract><cop>New York</cop><pub>Nature Publishing Group US</pub><pmid>24727651</pmid><doi>10.1038/nmeth.2928</doi><tpages>6</tpages><orcidid>https://orcid.org/0000-0002-2420-2593</orcidid><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; SpringerLink Journals; Nature |
| subjects | 14/35 631/136/1660 631/57/2265 631/80/2373 631/80/85 64/11 Analysis Animals Bioinformatics Biological Microscopy Biological Techniques Biomedical Engineering/Biotechnology Caenorhabditis elegans - embryology Caenorhabditis elegans - metabolism Caenorhabditis elegans Proteins - metabolism Cell physiology Cellular biology Dynamics Embryo, Nonmammalian Embryonic development Embryos Genetic aspects Green Fluorescent Proteins - metabolism Kinetics Life Sciences Membrane proteins Mobility Molecular Imaging Nematodes Photobleaching Physiological aspects Proteins Proteomics Surface Properties |
| title | Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos |
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