Interação entre IQGAP1 e actina mediado por CDC42 e RAC1
Ano de defesa: | 2010 |
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Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , , , |
Tipo de documento: | Dissertação |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Uberlândia
|
Programa de Pós-Graduação: |
Programa de Pós-graduação em Genética e Bioquímica
|
Departamento: |
Ciências Biológicas
|
País: |
BR
|
Palavras-chave em Português: | |
Área do conhecimento CNPq: | |
Link de acesso: | https://repositorio.ufu.br/handle/123456789/15823 |
Resumo: | IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which in turn activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound forms of Cdc42 and Rac1, which also bind IQGAP1. We report here the use of purified proteins for in vitro binding and actin polymerization assays, and of fluorescence resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions involving IQGAP1, N-WASP, and either Cdc42 or Rac1. In pyrene-actin assembly assays in the presence of N-WASP and the Arp2/3 complex, Cdc42 and IQGAP1 cooperatively stimulated actin filament nucleation, primarily by reducing the lag time before assembly Vmax was reached. Cooperativity reflected dose-dependent stimulation by Cdc42 of IQGAP1 binding to N-WASP. Rac1 and IQGAP1 behaved differently. At low Rac1, the two proteins cooperatively reduced the lag time before assembly Vmax was reached, but at high Rac1 Vmax was faster and reached more quickly for Rac1 alone than for either IQGAP1 alone, or the combination of Rac1 and IQGAP1. This negative cooperativity reflected dose-dependent inhibition by Rac1 of IQGAP1 binding to N-WASP. These results suggest that IQGAP1 interacts by distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. To address this possibility, FRET microscopy was used to study interactions of GFP-IQGAP1 with mOrange-Cdc42 versus mOrange-Rac1 in live MDCK cells. Robust FRET was observed for both donor/acceptor pairs at F-actin rich cell-cell margins, but the average intermolecular FRET distances closer for IQGAP1-Cdc42 than for IQGAP1-Rac1. The distinct interactions of IQGAP1 with Cdc42 versus Rac1 observed in vitro were thus recapitulated in live cells. |
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2016-06-22T18:43:43Z2010-09-212016-06-22T18:43:43Z2010-07-19PEREIRA, Rafael Luzes. Interação entre IQGAP1 e actina mediado por CDC42 e RAC1. 2010. 74 f. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2010.https://repositorio.ufu.br/handle/123456789/15823IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which in turn activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound forms of Cdc42 and Rac1, which also bind IQGAP1. We report here the use of purified proteins for in vitro binding and actin polymerization assays, and of fluorescence resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions involving IQGAP1, N-WASP, and either Cdc42 or Rac1. In pyrene-actin assembly assays in the presence of N-WASP and the Arp2/3 complex, Cdc42 and IQGAP1 cooperatively stimulated actin filament nucleation, primarily by reducing the lag time before assembly Vmax was reached. Cooperativity reflected dose-dependent stimulation by Cdc42 of IQGAP1 binding to N-WASP. Rac1 and IQGAP1 behaved differently. At low Rac1, the two proteins cooperatively reduced the lag time before assembly Vmax was reached, but at high Rac1 Vmax was faster and reached more quickly for Rac1 alone than for either IQGAP1 alone, or the combination of Rac1 and IQGAP1. This negative cooperativity reflected dose-dependent inhibition by Rac1 of IQGAP1 binding to N-WASP. These results suggest that IQGAP1 interacts by distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. To address this possibility, FRET microscopy was used to study interactions of GFP-IQGAP1 with mOrange-Cdc42 versus mOrange-Rac1 in live MDCK cells. Robust FRET was observed for both donor/acceptor pairs at F-actin rich cell-cell margins, but the average intermolecular FRET distances closer for IQGAP1-Cdc42 than for IQGAP1-Rac1. The distinct interactions of IQGAP1 with Cdc42 versus Rac1 observed in vitro were thus recapitulated in live cells.O citoesqueleto é um componente vital, tendo algumas funções básicas para o funcionamento normal e a manutenção das atividades celulares, como a emissão de pseudópodes, o transporte de vesículas e de outros componentes, a migração, entre outros. Estão envolvidos nesses processos microtúbulos, filamentos de actina, filamentos intermediários e várias proteínas associadas. Dentre essas proteínas, podemos citar as pertencentes à familia IQGAP. Elas são proteínas reguladoras da dinâmica de actina, mediadas por GTP e GDP. Três delas estão presentes em humanos, IQGAP1, IQGAP2 e IQGAP3. Essas proteínas são muito similares entre si e têm seus domínios e sequências primárias muito conservadas. A regulação e interações destas proteínas, com actina e pequenas GTPases também são descritas de maneira muito similar. A interação das proteínas IQGAPs com Cdc42, Rac1 e Rho é muito importante na regulação da dinâmica do filamento de actina, podendo acelerar a polimerização ou deprimir, dependendo da interação especificada. Podemos ver como elas estimulam a polimerização de actina, sozinhas ou na presença de IQGAP1. O processo de purificação de IQGAP1 nativo, de rins bovinos, foi elaborado para melhor observar a dinâmica desta proteína. Embora a ligação entre IQGAP1 e actina tenha se mostrado muito forte e resistente a diversos processos de dissociação, mostrou-se sensível à concentração de NaCl. Dentro deste trabalho nosso principal objetivo foi estudar a influência que a interação entre Rac1 e Cdc42 com IQGAP1 exercem na dinâmica do filamento de actina e, mais especificamente, avaliar a influência, no filamento de actina, das interações entre IQGAP1 e Rac1 e IQGAP1 e Cdc42, comparativamente, alem de desenvolver um método de purificação de IQGAP1 nativo.Mestre em Genética e Bioquímicaapplication/pdfporUniversidade Federal de UberlândiaPrograma de Pós-graduação em Genética e BioquímicaUFUBRCiências BiológicasProteínasCitoesqueletoIQGAP1ActinaRac1Cdc42CNPQ::CIENCIAS BIOLOGICAS::GENETICAInteração entre IQGAP1 e actina mediado por CDC42 e RAC1info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisCameron, Luiz Claudiohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782703P3Hamaguchi, Améliahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4793225J6Bassini, Adrianahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4758169E6Coelho, Milton Vieirahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785825A8Provance Junior, David Williamhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4460795J4http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4251631Y7Pereira, Rafael Luzesinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFUTHUMBNAILDiss Rafael.pdf.jpgDiss Rafael.pdf.jpgGenerated Thumbnailimage/jpeg1191https://repositorio.ufu.br/bitstream/123456789/15823/3/Diss%20Rafael.pdf.jpg1823749c2718820a28265d1f831deb85MD53ORIGINALDiss Rafael.pdfapplication/pdf1557715https://repositorio.ufu.br/bitstream/123456789/15823/1/Diss%20Rafael.pdfd006e28e70eb86163381127ccec74277MD51TEXTDiss Rafael.pdf.txtDiss Rafael.pdf.txtExtracted texttext/plain98302https://repositorio.ufu.br/bitstream/123456789/15823/2/Diss%20Rafael.pdf.txt858111b65ae29d9ac064aa5fb476b83dMD52123456789/158232016-06-23 04:20:47.673oai:repositorio.ufu.br:123456789/15823Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2016-06-23T07:20:47Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false |
dc.title.por.fl_str_mv |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 |
title |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 |
spellingShingle |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 Pereira, Rafael Luzes Proteínas Citoesqueleto IQGAP1 Actina Rac1 Cdc42 CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
title_short |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 |
title_full |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 |
title_fullStr |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 |
title_full_unstemmed |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 |
title_sort |
Interação entre IQGAP1 e actina mediado por CDC42 e RAC1 |
author |
Pereira, Rafael Luzes |
author_facet |
Pereira, Rafael Luzes |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Cameron, Luiz Claudio |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782703P3 |
dc.contributor.referee1.fl_str_mv |
Hamaguchi, Amélia |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4793225J6 |
dc.contributor.referee2.fl_str_mv |
Bassini, Adriana |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4758169E6 |
dc.contributor.referee3.fl_str_mv |
Coelho, Milton Vieira |
dc.contributor.referee3Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785825A8 |
dc.contributor.referee4.fl_str_mv |
Provance Junior, David William |
dc.contributor.referee4Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4460795J4 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4251631Y7 |
dc.contributor.author.fl_str_mv |
Pereira, Rafael Luzes |
contributor_str_mv |
Cameron, Luiz Claudio Hamaguchi, Amélia Bassini, Adriana Coelho, Milton Vieira Provance Junior, David William |
dc.subject.por.fl_str_mv |
Proteínas Citoesqueleto IQGAP1 Actina Rac1 Cdc42 |
topic |
Proteínas Citoesqueleto IQGAP1 Actina Rac1 Cdc42 CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
description |
IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which in turn activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound forms of Cdc42 and Rac1, which also bind IQGAP1. We report here the use of purified proteins for in vitro binding and actin polymerization assays, and of fluorescence resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions involving IQGAP1, N-WASP, and either Cdc42 or Rac1. In pyrene-actin assembly assays in the presence of N-WASP and the Arp2/3 complex, Cdc42 and IQGAP1 cooperatively stimulated actin filament nucleation, primarily by reducing the lag time before assembly Vmax was reached. Cooperativity reflected dose-dependent stimulation by Cdc42 of IQGAP1 binding to N-WASP. Rac1 and IQGAP1 behaved differently. At low Rac1, the two proteins cooperatively reduced the lag time before assembly Vmax was reached, but at high Rac1 Vmax was faster and reached more quickly for Rac1 alone than for either IQGAP1 alone, or the combination of Rac1 and IQGAP1. This negative cooperativity reflected dose-dependent inhibition by Rac1 of IQGAP1 binding to N-WASP. These results suggest that IQGAP1 interacts by distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. To address this possibility, FRET microscopy was used to study interactions of GFP-IQGAP1 with mOrange-Cdc42 versus mOrange-Rac1 in live MDCK cells. Robust FRET was observed for both donor/acceptor pairs at F-actin rich cell-cell margins, but the average intermolecular FRET distances closer for IQGAP1-Cdc42 than for IQGAP1-Rac1. The distinct interactions of IQGAP1 with Cdc42 versus Rac1 observed in vitro were thus recapitulated in live cells. |
publishDate |
2010 |
dc.date.available.fl_str_mv |
2010-09-21 2016-06-22T18:43:43Z |
dc.date.issued.fl_str_mv |
2010-07-19 |
dc.date.accessioned.fl_str_mv |
2016-06-22T18:43:43Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
PEREIRA, Rafael Luzes. Interação entre IQGAP1 e actina mediado por CDC42 e RAC1. 2010. 74 f. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2010. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufu.br/handle/123456789/15823 |
identifier_str_mv |
PEREIRA, Rafael Luzes. Interação entre IQGAP1 e actina mediado por CDC42 e RAC1. 2010. 74 f. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2010. |
url |
https://repositorio.ufu.br/handle/123456789/15823 |
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por |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Uberlândia |
dc.publisher.program.fl_str_mv |
Programa de Pós-graduação em Genética e Bioquímica |
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UFU |
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BR |
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Ciências Biológicas |
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Universidade Federal de Uberlândia |
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