Analyses of mitotic nuclear pore complex dynamics in Aspergillus nidulans

Regulated cell division is critical for normal development of multicellular organisms and abnormalities in cell division can lead to cancer and other diseases. Our understanding of many aspects of cell division is still in its infancy. For instance, in higher eukaryotes the nucleus breaks down and becomes reassembled during every cell division with amazingly high fidelity. However, very little is known about how this massive reorganization is achieved. The nucleus is bound by a nuclear envelope (NE) decorated with many nuclear pore complexes (NPCs). These NPCs form aqueous channels across the NE to allow communication between the nucleus and cytoplasm. Previously, we demonstrated that the model filamentous fungus Aspergillus nidulans partially disassembles its NPCs to allow cell cycle regulators and microtubules to gain access to the mitotic chromatin. During this process, a core NPC structure containing the An-Nup84-120 subcomplex, An-Nup170, An-Gle1, and two transmembrane nucleoporins (Nups), An-Ndc1 and An-Pom152 remain in the NE whilst all peripheral Nups disperse. The minimal NPC core components form a scaffold across the NE to anchor the core NPC structure and to recruit dispersed Nups back to the NE during exit from mitosis. The aim of this study was to understand how NPC structure is maintained and assembled by studying the composition and function of the NPC core components in A. nidulans.;To further understand how NPC structure is maintained, it is necessary to define the protein-protein interaction networks between Nups. In this study, an efficient S-Tag affinity purification protocol has been established allowing the identification of protein complexes and their post-translational modifications. Applying this technique, two novel fungal Nups, termed An-Nup37 and An-ELYS, were identified through the affinity purifications of components of the major core subcomplex, the An-Nup84-120 complex. Like other components of the An-Nup84-120 complex, An-Nup37 and An-ELYS remain at the NPC throughout interphase but partially concentrate to the spindle pole bodies (SPBs) during mitosis. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE suggesting the An-Nup84-120 complex might function with membrane. Supporting this, mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol and high temperature. Most informatively, cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152 and An-Pom34) are viable but An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. This study indicates that the core An-Nup84-120 subcomplex and transmembrane Nups cooperate to perform essential roles at the NPC membrane and provide resistance to increasing membrane fluidity.;Studies in A. nidulans and vertebrates have suggested that NPC reassembly is a stepwise process. It is generally accepted that the vertebrate Nup107-160 complex (homologue of the An-Nup84-120 complex) is the first subcomplex required to initiate NPC assembly. However, we have demonstrated that the An-Nup84-120 complex can be further disassembled and reassembled from the mitotic NPC core structure. This result suggests that An-Nup170, An-Gle1, and transmembrane Nups that remain at the NE may play an earlier role in NPC assembly. Unlike transmembrane Nups, An-Nup170 and An-Gle1 are both essential genes and my preliminary data suggested that An-Nup170 may play an earlier role than An-Gle1 in NPC reassembly. Thus, to uncover the mechanism that initiates NPC assembly, An-Nup170 was characterized in more detail. This work demonstrates that An-Nup170 is required for NPC assembly in both interphase and mitosis as in the absence of An-Nup170, all Nups tested become mis-localized. In addition, nuclear organization and segregation are defective in DeltaAn-nup170 cells suggesting that An-Nup170 is a critical link between the NE and chromatin. Further characterization of An-Nup170 and its relationship with chromatin by live cell imaging revealed that An-Nup170 and other core Nups initiate their assembly from the chromatin that is near the SPBs.;In conclusion, this dissertation has established powerful tools that have helped advance the field. Appling these developed protocols, this work has defined the redundant function of non-essential core NPC proteins in maintaining pore membrane stability and characterized the essential role of An-Nup170 in NPC assembly.