As vias de Cdc42/N-WASP e Rac1/WAVE2 na dinâmica de actina durante a invasão celular pelos amastigotas extracelulares de Trypanosoma cruzi
Host cell invasion by extracellular amastigotes (EA) of Trypanosoma cruzi is highly dependent on actin cytoskeleton of host cells whose regulatory cellular mechanisms are still poorly understood. Cdc42 and Rac1 GTPases are key mediators of the actin cytoskeleton promoting Arp2/3 complex-dependent ac...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | Brasil |
| Institución: | Universidade Federal de São Paulo (UNIFESP) |
| Repositorio: | Repositório Institucional da UNIFESP |
| Idioma: | portugués |
| OAI Identifier: | oai:repositorio.unifesp.br:11600/47998 |
| Acceso en línea: | https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=4231800 http://repositorio.unifesp.br/handle/11600/47998 |
| Access Level: | acceso abierto |
| Palabra clave: | Trypanosoma cruzi Extracellular amastigotes Host cell invasion Actin Gtpases Amastigotas extracelulares Invasão celular Actina |
| Sumario: | Host cell invasion by extracellular amastigotes (EA) of Trypanosoma cruzi is highly dependent on actin cytoskeleton of host cells whose regulatory cellular mechanisms are still poorly understood. Cdc42 and Rac1 GTPases are key mediators of the actin cytoskeleton promoting Arp2/3 complex-dependent actin polymerization via activation of their effector proteins, N-WASP and WAVE2, respectively. The aim of this study was to evaluate the participation Cdc42/N-WASP and Rac1/WAVE2 signaling pathways in actin dynamics during EA internazalization in HeLa cells. Using live-cell imaging in confocal microscope it was observed that Cdc42 and Rac1 are recruited to and colocalize with actin during whole period of EA internalisation. GTPases recruitment was sustained in groups expressing active (CA) or inactive (DN) mutant isoforms when compared to native isoforms (WT). When the invasion ability was compared to control groups, the expression of Rac1 CA and Cdc42 WT increased the number of internalized parasites while Rac1 DN and Cdc42 CA expression reduced it. Additionally, the invasion of EAs is inhibited in cells depleted for Rac1 and also recruitment assays using live cells showed delayed invasion despite no effective reduction in amount of polymerized actin at EA invasion sites. For cells depleted for Cdc42 it was observed inhibition of EA internalization in some experiments, but no inhibition in others; live-cell imaging assays also revealed no delay in actin recruitment in this group. In cells depleted for N-WASP and WAVE2 proteins it was also observed inhibition and delay in the internalization without reduction in actin polymerization. Both proteins were also recruited to and colocalized with actin in EA invasion sites. Finally, depletion of four proteins studied did not affect the density or morphology of membrane projections mobilized by AEs as observed by scanning electron microscopy. The overall result confirms the participation of Cdc42/N-WASP and Rac1/WAVE2 signaling pathways in actin dynamics during EA host cell invasion and encourage the study of other proteins possibly cooperating in these pathways. |
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