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| Author(s): |
Bernardo Pereira Moreira
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2016-12-16 |
| Examining board members: |
Munira Muhammad Abdel Baqui;
Angela Kaysel Cruz;
Juliana Lopes Rangel Fietto;
Sergio Schenkman;
Luiz Ricardo Orsini Tosi
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| Advisor: | Munira Muhammad Abdel Baqui |
| Abstract | |
Trypanosoma brucei, the causative agent of sleeping sickness or Human African Trypanosomiasis (HAT), has been used as experimental model for cellular, biochemical and molecular studies. This is an extracellular protozoan parasite that has a single flagellum and a highly stable cytoskeleton, responsible for many cellular processes such as motility, morphology, infectivity and cell division. In Trypanosomatidae family, a novel class of High Molecular Weight Proteins (HMWPs; 500-3500 kDa) has been described, which besides their structural role, may play a role in cytoskeleton organization and regulation. Trypanosomatids cytoskeletons contain abundant HMWPs, but many of their biological functions are still unclear. Here, we aimed to describe the cellular and molecular properties of these proteins, and to determine their functional roles in the parasite biology. SDS-PAGE was used to analyze T. brucei cytoskeletons. Also, the HMWP bands were injected in mouse in order to produce polyclonal antibodies, which were used as a molecular tool in Western Blotting (WB) and immunofluorescence analysis. Here we show that the high molecular weight proteins are located on essential structures for the regulation of the cytoskeleton such as the flagellar adhesion zone (FAZ) and the tripartite complex, which connects the flagellum to the kinetoplast. Detergent-extracted cytoskeletons were also analyzed by gradient SDS-PAGE and the HMWP bands were sent to mass spectrometry analysis. We able to identify 19 new high molecular weight proteins most of which are uncharacterized so far in T. brucei. Here, we report the characterization of two giant proteins: FAZ10 and Tb927.8.3540. To investigate their biological roles we used a model of gene silencing by RNA interference (RNAi). Our data showed that FAZ10 is an essential giant cytoskeletal protein in both procyclic and bloodstream parasite life cycle stages, since its depletion led to defects in cell morphogenesis, flagellum attachment and kinetoplast and nucleus positioning. More importantly, ablation of FAZ10 impaired the timing and placement of the cleavage furrow during cytokinesis, resulting in premature or asymmetrical cell division. In turn, the Tb927.8.3540 protein is responsible for the stabilization and regulation of the microtubule cytoskeleton as well as the flagellum and FAZ. In its absence, cells acquire a spherical shape together with an internalized flagellum. In both cases, the resulting phenotype results in inhibition of cell proliferation and accumulation of multinucleated cells in culture. The functional study of the giant proteins revealed that they have a central role in morphogenesis of T. brucei, acting directly on the organization and regulation of cytoskeletal structures, such as the flagellum and the FAZ. (AU) | |
| FAPESP's process: | 12/22129-2 - Functional characterization of giant protein in Trypanosoma brucei |
| Grantee: | Bernardo Pereira Moreira |
| Support Opportunities: | Scholarships in Brazil - Doctorate |