{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T00:29:13Z","timestamp":1777508953349,"version":"3.51.4"},"reference-count":17,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2015,3,9]],"date-time":"2015-03-09T00:00:00Z","timestamp":1425859200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"NIH grants","award":["NS051746, HL101871."],"award-info":[{"award-number":["NS051746, HL101871."]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2015,6]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Experiments using live cell 3\u2010color F\u00f6rster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3\u2010color FRET data is demonstrated, extending the analysis beyond the customary energy\u2010transfer efficiency (<jats:italic>E<\/jats:italic>%) calculations. MDCK cells were transfected for coexpression of Teal\u2010N\u2010WASP\/Venus\u2010IQGAP1\/mRFP1\u2010Rac1, Teal\u2010N\u2010WASP\/Venus\u2010IQGAP1\/mRFP1\u2010Cdc42, CFP\u2010Rac1\/Venus\u2010IQGAP1\/mCherry\u2010actin, or CFP\u2010Cdc42\/Venus\u2010IQGAP1\/mCherry\u2010actin, and with single\u2010label equivalents for spectral bleedthrough correction. Using confirmed <jats:italic>E<\/jats:italic>% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP\u2010Rac1:Venus\u2010IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP\u2010Cdc42:Venus\u2010IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1\u2010Cdc42 or mRFP1\u2010Rac1, respectively, promoted or suppressed the association of Teal\u2010N\u2010WASP with Venus\u2010IQGAP1. These 3\u2010color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N\u2010WASP with IQGAP1. In addition, this study emphasizes the power of 3\u2010color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. \u00a9 2015 International Society for Advancement of Cytometry<\/jats:p>","DOI":"10.1002\/cyto.a.22651","type":"journal-article","created":{"date-parts":[[2015,3,9]],"date-time":"2015-03-09T20:35:49Z","timestamp":1425933349000},"page":"580-588","source":"Crossref","is-referenced-by-count":12,"title":["Three\u2010color confocal F\u00f6rster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells"],"prefix":"10.1002","volume":"87","author":[{"given":"Horst","family":"Wallrabe","sequence":"first","affiliation":[{"name":"Department of Biology W.M. Keck Center for Cellular Imaging University of Virginia Charlottesville Virginia"},{"name":"Department of Biology University of Virginia Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuansheng","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Biology W.M. Keck Center for Cellular Imaging University of Virginia Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaolan","family":"Fang","sequence":"additional","affiliation":[{"name":"Department of Biology University of Virginia Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ammasi","family":"Periasamy","sequence":"additional","affiliation":[{"name":"Department of Biology W.M. Keck Center for Cellular Imaging University of Virginia Charlottesville Virginia"},{"name":"Department of Biology University of Virginia Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"George S.","family":"Bloom","sequence":"additional","affiliation":[{"name":"Department of Biology University of Virginia Charlottesville Virginia"},{"name":"Department of Cell Biology University of Virginia Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2015,3,9]]},"reference":[{"key":"e_1_2_5_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/cm.21146"},{"key":"e_1_2_5_3_1","first-page":"19","article-title":"Imaging protein molecules using FRET and FLIM microscopy","volume":"16","author":"Jares\u2010Erijman EA","year":"2003","journal-title":"Curr Opin Biotech"},{"key":"e_1_2_5_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/cphc.201000664"},{"key":"e_1_2_5_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2004.12.002"},{"key":"e_1_2_5_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(03)75126-1"},{"key":"e_1_2_5_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-019517720-6.50016-X"},{"key":"e_1_2_5_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1046-2023(02)00283-9"},{"key":"e_1_2_5_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2010.06.004"},{"key":"e_1_2_5_10_1","doi-asserted-by":"publisher","DOI":"10.1117\/12.906432"},{"key":"e_1_2_5_11_1","doi-asserted-by":"publisher","DOI":"10.1117\/1.3227036"},{"key":"e_1_2_5_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.26.15362"},{"key":"e_1_2_5_13_1","first-page":"89481J","article-title":"Regulatory conformational changes of the epsilon subunit in single FRET\u2010labeled FF\u2010ATP synthase","volume":"8948","author":"Duncan TM","year":"2014","journal-title":"Proc Soc Photo Opt Instrum Eng"},{"key":"e_1_2_5_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/jp3064273"},{"key":"e_1_2_5_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja408767p"},{"key":"e_1_2_5_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2012.07.026"},{"key":"e_1_2_5_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/psc.2590"},{"key":"e_1_2_5_18_1","doi-asserted-by":"publisher","DOI":"10.1364\/OE.20.026806"}],"container-title":["Cytometry Part A"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fcyto.a.22651","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.a.22651","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,13]],"date-time":"2023-09-13T03:21:44Z","timestamp":1694575304000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cyto.a.22651"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2015,3,9]]},"references-count":17,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2015,6]]}},"alternative-id":["10.1002\/cyto.a.22651"],"URL":"https:\/\/doi.org\/10.1002\/cyto.a.22651","archive":["Portico"],"relation":{},"ISSN":["1552-4922","1552-4930"],"issn-type":[{"value":"1552-4922","type":"print"},{"value":"1552-4930","type":"electronic"}],"subject":[],"published":{"date-parts":[[2015,3,9]]}}}