Functional study of Nucleoporins Nup88 and Nup214 during Drosophila Development

Sammanfattning: More than a million macromolecules per minute pass through the nuclear envelope of a eukaryotic cell. An important challenge is to understand how the NPC coordinate efficiently the bi-directional trafficking necessary for the basal cellular activities and the rapid translocation of regulatory proteins in response to signaling. In higher eukaryotes each NPC contains approximately 30 different proteins. Some of these have been mapped to distinct parts of the NPC and shown to interact with transport receptors. However, it is not clear whether the different nucleoporins may directly participate in distinct nuclear transport events leading to regulatory changes of gene expression, or if they all may be involved in providing an elaborate but passive channel for general nuclear entry.The work presented in this thesis begins with the characterization of Drosophila nucleoporin Nup88, which together with the nucleoporin Nup214 forms a complex in the cytoplasmic filaments of the NPC. Unlike Nup214, Nup88 has a cell specific expression suggesting that different cells types have selective requirement of Nup88s intrinsic function during animal development. Nup88 and Nup214 are selectively required for REL protein translocation upon signaling. In nup88 mutants the upstream signaling cascade leading to the degradation of IkB homolog Cactus is functional however, Dorsal and Dif remain cytoplasmic in both mutants rendering inactive their immune response.We showed that both nucleoporins Nup88 and Nup214 are interdependent for their localization at the nuclear envelope. In the absence of Nup214, Nup88 becomes degraded and Nup88 is necessary for localizing Nup214 to the NPC. The complex is required for tethering CRM1 on the nuclear envelope and its major function is to attenuate nuclear export. Immunoprecipitations from embryonic extract suggest that the affinity of the complex for CRM1 could be modulated during embryo development. Reduction of the gene dosage on either Nup88 or Nup214 allow the progression of CRM1 mutants through several developmental stages suggesting that the barrier for protein export provided by anchoring a pool of CRM1 to the Nup88/214 complex has important functions in Drosophila development.Finally, detailed analysis of Nup88, Nup214 and CRM1 mutants reveals that the REL protein translocation and the activation of the innate immune responses are dependent on the inhibitory action of the Nup88/Nup214 complex on CRM1 export.Our results suggest a new mechanism of regulation through NPC, where dynamic interplay between nucleoporin complexes can accommodate transport of signaling effectors in response to physiological responses.