Understanding RanGTP dependent microtubule assembly : Idenification of DnaJB6 as a RanGTP regulated factor involved in microtubule organization during mitosis
Three microtubule (MT) assembly pathways participate in the assembly of the bipolar spindle: the centrosomal pathway, the augmin dependent amplification pathway and the RanGTP/chromosome dependent pathway. To form the spindle, all these MTs are organized by various classes of motor proteins into two...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/664169 |
| Acceso en línea: | http://hdl.handle.net/10803/664169 |
| Access Level: | acceso abierto |
| Palabra clave: | Microtubule DnaJB6 Ran Mitosis Spindle Microtubul Fus 576 |
| Sumario: | Three microtubule (MT) assembly pathways participate in the assembly of the bipolar spindle: the centrosomal pathway, the augmin dependent amplification pathway and the RanGTP/chromosome dependent pathway. To form the spindle, all these MTs are organized by various classes of motor proteins into two interdigitating antiparallel arrays with their minus ends focused at the spindle poles. This focusing activity is provided by the minus-end directed motor proteins Dynein-Dynactin and HSET. Spindle assembly can occur in the absence of centrosomes indicating that the RanGTP and augmin dependent pathways are sufficient. The RanGTP pathway can be studied in Xenopus laevis egg extracts. Addition of RanGTP to these extracts triggers a dynamic process of MT nucleation, stabilization and organization into asters and mini spindles. To obtain a global picture of the RanGTP pathway we used a proteomics approach and determined the interactome of the RanGTP-MTs that consists of 1263 proteins. Moreover we have analyzed the changes in this proteome to try to correlate them with the change in MT dynamic and organization observed upon different time of incubation of the egg extract with RanGTP. Although the composition of the proteome did not change, we found different patterns of recruitment for various protein groups. The proteome includes most of the known RanGTP regulated factors in mitosis and significantly overlaps with previously published spindle and taxol-MT proteomes. In addition it contains a large number of other proteins with described or undescribed functions in various cellular processes. We used this proteome to identify novel putative RanGTP regulated spindle assembly factors (SAFs). We identified DnaJB6 as a RanGTP regulated protein involved in spindle assembly. We found that it interacts with dynactin p150 in a RanGTP dependent manner specifically in M-phase. We show that DnaJB6 favors the stabilization of the Dynactin complex specifically in mitosis, regulating the activity of Dynein-Dynactin complex in bipolar spindle assembly and MT focusing at the spindle poles. |
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