Dokument

Summary:
This work presents findings on dynamic nuclear pore behaviour in interphase nuclei and in the “open” mitosis in Ustilago maydis. Proteins likely to function in the nuclear pore complexes (NPCs) are identified in the U. maydis genome by bioinformatic search. Of these, five nucleoporins are tagged with GFP and observed microscopically for their localization in interphase and mitosis. One of the stably incorporated nucleoporins, Nup107, is analyzed in more detail. Nup107 appears to be essential in U. maydis, with depletion of the protein causing NPC clustering at the nuclear envelope. The initial observation reveals that NPCs in U. maydis are motile. The movement of NPCs in interphase nuclei has been reported in budding yeast, but a detailed analysis of the underlying mechanism and of the biological significance of the phenomenon is missing. In contrast to the studies in budding yeast, which assumed diffusion as cause for NPC motility, this work in U. maydis finds directed motility of NPCs. The directed motility is energy-dependent and proceeds in two motility types. Type 1 depends on the microtubule cytoskeleton. Dynein is the driving force behind MINUS end directed movement. NPC type 2 motility is abolished when active transcription is inhibited, but cannot be directly correlated with chromatin movement. FRAP experiments demonstrate that type 1 motility is necessary to equally distribute NPCs across the nuclear envelope. Equal distribution is required to ensure efficient protein expression of a reporter from an inducible promoter. Following the GFP-tagged nucleoporins from different parts of the NPC structure into mitosis, it appears that NPCs are still assembled in prophase. A tendency of NPCs to accumulate at the tip of the elongated prophase is observed, but the function of NPCs in chromosome migration appears doubtful. At the end of prophase, NPCs disassemble into subcomplexes and disperse in different locations, with the Nup107-160 subcomplex associating with chromatin in metaphase. Complete nuclear envelope (NE) removal is not necessary for NPC disassembly and association of Nup107 with DNA. In contrast to findings in vertebrates, the Nup107-160 subcomplex does not associate with kinetochores in metaphase. In anaphase, the Nup107-160 subcomplex shifts its position to the leading outside edges of the chromatin. Nucleoporins accumulate at the NEs in telophase in a step-wise manner, and nuclear import starts after all nucleoporins investigated are assembled at the nuclei. Inhibitor studies to investigate the role of the cytoskeleton cannot fully explain the involvement of actin and microtubules in NE reassembly. The results from these observations on NPC motility in interphase nuclei open a new view on basic principles of ensuring efficient protein expression. The findings on NPC disassembly in mitosis place U. maydis close to the vertebrate situation, suggesting a more ancient origin for the “open” mitosis.

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