Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy
Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR-FCS makes possible the characterization...
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| Veröffentlicht in: | Biophysical journal 2006-11, Vol.91 (9), p.3456-3464 |
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| creator | Ohsugi, Yu Saito, Kenta Tamura, Mamoru Kinjo, Masataka |
| description | Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR-FCS makes possible the characterization of reversible association and dissociation rates between fluorescent ligands and their receptors in supported phospholipid bilayers. Here, for the first time, we extend TIR-FCS to a cellular application for measuring the lateral diffusion of a membrane-binding fluorescent protein, farnesylated EGFP, on the plasma membranes of cultured HeLa and COS7 cells. We detected two kinds of diffusional motion—fast three-dimensional diffusion (
D
1) and much slower two-dimensional diffusion (
D
2), simultaneously. Conventional FCS and single-molecule tracking confirmed that
D
1 was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and
D
2 was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells. |
| doi_str_mv | 10.1529/biophysj.105.074625 |
| format | Article |
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D
1) and much slower two-dimensional diffusion (
D
2), simultaneously. Conventional FCS and single-molecule tracking confirmed that
D
1 was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and
D
2 was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells.</description><identifier>ISSN: 0006-3495</identifier><identifier>EISSN: 1542-0086</identifier><identifier>DOI: 10.1529/biophysj.105.074625</identifier><identifier>PMID: 16891361</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Biophysics ; Cell Membrane - metabolism ; Cells ; Cercopithecus aethiops ; COS Cells ; Feasibility Studies ; Fluorescence ; HeLa Cells ; Humans ; Membrane Proteins - metabolism ; Membranes ; Microscopy, Confocal - methods ; Molecular Probe Techniques ; Protein Transport - physiology ; Proteins ; Spectrometry, Fluorescence - methods ; Spectroscopy, Imaging, Other Techniques ; Spectrum analysis</subject><ispartof>Biophysical journal, 2006-11, Vol.91 (9), p.3456-3464</ispartof><rights>2006 The Biophysical Society</rights><rights>Copyright Biophysical Society Nov 1, 2006</rights><rights>Copyright © 2006, Biophysical Society 2006</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c623t-d68e31c689fdea6e2021ecb67607e0e8fca1a1f46fbed6a54be2c174567ded3b3</citedby><cites>FETCH-LOGICAL-c623t-d68e31c689fdea6e2021ecb67607e0e8fca1a1f46fbed6a54be2c174567ded3b3</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/PMC1614500/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://dx.doi.org/10.1529/biophysj.105.074625$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,724,777,781,882,3537,27905,27906,45976,53772,53774</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16891361$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ohsugi, Yu</creatorcontrib><creatorcontrib>Saito, Kenta</creatorcontrib><creatorcontrib>Tamura, Mamoru</creatorcontrib><creatorcontrib>Kinjo, Masataka</creatorcontrib><title>Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy</title><title>Biophysical journal</title><addtitle>Biophys J</addtitle><description>Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR-FCS makes possible the characterization of reversible association and dissociation rates between fluorescent ligands and their receptors in supported phospholipid bilayers. Here, for the first time, we extend TIR-FCS to a cellular application for measuring the lateral diffusion of a membrane-binding fluorescent protein, farnesylated EGFP, on the plasma membranes of cultured HeLa and COS7 cells. We detected two kinds of diffusional motion—fast three-dimensional diffusion (
D
1) and much slower two-dimensional diffusion (
D
2), simultaneously. Conventional FCS and single-molecule tracking confirmed that
D
1 was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and
D
2 was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells.</description><subject>Animals</subject><subject>Biophysics</subject><subject>Cell Membrane - metabolism</subject><subject>Cells</subject><subject>Cercopithecus aethiops</subject><subject>COS Cells</subject><subject>Feasibility Studies</subject><subject>Fluorescence</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Membrane Proteins - metabolism</subject><subject>Membranes</subject><subject>Microscopy, Confocal - methods</subject><subject>Molecular Probe Techniques</subject><subject>Protein Transport - physiology</subject><subject>Proteins</subject><subject>Spectrometry, Fluorescence - methods</subject><subject>Spectroscopy, Imaging, Other Techniques</subject><subject>Spectrum analysis</subject><issn>0006-3495</issn><issn>1542-0086</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNp9kc1u1DAUhS1ERYfCEyAhiwW7TG0ndiYLkGBEodJUIChry7FvWo88dmonI-UReGscZlp-Fqxs-X73-N5zEHpByZJy1py3NvS3U9ouKeFLUleC8UdoQXnFCkJW4jFaEEJEUVYNP0VPU9oSQhkn9Ak6pWLV0FLQBfqxUQNE5fBVaK2zw4RDh69g10bloXhvvbH-Bn-JYQDrE7Yeb-x-flqDcymTKo0RDG4nfB2GrHPps57Pl6_QOdCDDR5fuDFESBq8BrwOMYJTvwrf-kzEkHTop2fopFMuwfPjeYa-X3y4Xn8qNp8_Xq7fbQotWDkURqygpDov0BlQAhhhFHQrakFqILDqtKKKdpXoWjBC8aoFpmldcVEbMGVbnqG3B91-bHdg8lBD3l_20e5UnGRQVv5d8fZW3oS9pIJWnJAs8PooEMPdCGmQO5t3cy47FsYk82i8IZxl8NU_4DaMszdJMspr2pTNDJUHSGcfUoTuYRJK5JyzvM85P3B5yDl3vfxzid89x2Az8OYAQLZybyHKpO3sv7Exey5NsP_94Cc_d7_e</recordid><startdate>20061101</startdate><enddate>20061101</enddate><creator>Ohsugi, Yu</creator><creator>Saito, Kenta</creator><creator>Tamura, Mamoru</creator><creator>Kinjo, Masataka</creator><general>Elsevier Inc</general><general>Biophysical Society</general><scope>6I.</scope><scope>AAFTH</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>3V.</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AF</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M2P</scope><scope>M7P</scope><scope>MBDVC</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>S0X</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20061101</creationdate><title>Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy</title><author>Ohsugi, Yu ; Saito, Kenta ; Tamura, Mamoru ; Kinjo, Masataka</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c623t-d68e31c689fdea6e2021ecb67607e0e8fca1a1f46fbed6a54be2c174567ded3b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Animals</topic><topic>Biophysics</topic><topic>Cell Membrane - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biophysical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ohsugi, Yu</au><au>Saito, Kenta</au><au>Tamura, Mamoru</au><au>Kinjo, Masataka</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy</atitle><jtitle>Biophysical journal</jtitle><addtitle>Biophys J</addtitle><date>2006-11-01</date><risdate>2006</risdate><volume>91</volume><issue>9</issue><spage>3456</spage><epage>3464</epage><pages>3456-3464</pages><issn>0006-3495</issn><eissn>1542-0086</eissn><abstract>Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR-FCS makes possible the characterization of reversible association and dissociation rates between fluorescent ligands and their receptors in supported phospholipid bilayers. Here, for the first time, we extend TIR-FCS to a cellular application for measuring the lateral diffusion of a membrane-binding fluorescent protein, farnesylated EGFP, on the plasma membranes of cultured HeLa and COS7 cells. We detected two kinds of diffusional motion—fast three-dimensional diffusion (
D
1) and much slower two-dimensional diffusion (
D
2), simultaneously. Conventional FCS and single-molecule tracking confirmed that
D
1 was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and
D
2 was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>16891361</pmid><doi>10.1529/biophysj.105.074625</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Biophysical journal, 2006-11, Vol.91 (9), p.3456-3464 |
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| source | MEDLINE; Cell Press Free Archives; Elsevier ScienceDirect Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central |
| subjects | Animals Biophysics Cell Membrane - metabolism Cells Cercopithecus aethiops COS Cells Feasibility Studies Fluorescence HeLa Cells Humans Membrane Proteins - metabolism Membranes Microscopy, Confocal - methods Molecular Probe Techniques Protein Transport - physiology Proteins Spectrometry, Fluorescence - methods Spectroscopy, Imaging, Other Techniques Spectrum analysis |
| title | Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy |
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