Phosphoproteomics of Nuclear Pore Disassembly and Reassembly during Mitosis
EMSL Project ID
40291
Abstract
Eukaryotic cells segregate DNA equally to daughter cells during mitosis. Although mitosis is well characterized, its regulation is still poorly understood. The most extensive and least understood mitotic process is nuclear breakdown which includes the disassembly of nuclear pore complexes (NPCs), massive protein assemblies which act as conduits through the nuclear envelope. Because nuclear breakdown does not occur in model yeasts it cannot be studied using these powerful model systems. However, my lab has discovered that in the model filamentous fungus Aspergillus nidulans aspects of nuclear breakdown occur during mitosis. Most importantly, A. nidulans partially disassembles its NPCs during mitosis. We have additionally shown that NPC disassembly requires the activity of two mitotic protein kinases, Cdk1 and NIMA and genetic and biochemical evidence indicates that phosphorylation of several NPC proteins is important for NPC disassembly. Importantly, ectopic induction of the NIMA kinase is sufficient to promote NPC disassembly, even in cells arrested in S-phase of the cell cycle. These findings have provided new concepts regarding how closed mitosis is regulated and form the basis for this application.As a well developed model genetic and biochemical system A. nidulans provides many experimental advantages. Its genome has been sequenced and highly efficient gene manipulations by targeted gene replacements are routine as are large scale affinity purifications of endogenously tagged proteins. Importantly, we have recently affinity purified to homogeneity all known (29 proteins) NPC proteins from cell cycle staged A. nidulans cells and used mass spectroscopy to define all protein complexes of the NPC and how these protein complexes change from G2 upon entry into mitosis. Biochemical analysis of the purified Nups has identified several NPC proteins that are phosphorylated during mitosis. Our aim for this proposal is to define mitotically phosphorylated Nups, their sites of phosphorylation and how these change during mitosis. We plan to purify NPC protein complexes that we have found to be mitotically phosphorylated from cell cultures mitotically synchronized or arrested at different stages of mitosis. Key to this is our unique ability to inactivate either the Cdk1 or NIMA kinases causing a G2 arrest and then synchronously active the respective kinase and biochemically follow the subsequent synchronous progression through mitosis. Coupled with the state-of-the-art high end mass spectroscopy analysis available at the EMBL this will allow the phosphoproteome of the mitotic NPC to be defined. This data will enable us to critically test the in vivo function of NPC protein phosphorylation during mitotic NPC disassembly and reassembly and additionally uncover the mitotic kinases responsible.
We expect our analysis will advance our understanding of how a massive multi protein structure can be dismantled and reassembled in a highly regulated and sequential manner. The findings are expected to have a major impact on our understanding of the mitotic regulation of the NPC. The proposed research fits well with the EMSL Science Theme of determining the dynamics of protein composition or localization and their assembly into multiprotein complexes.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2010-10-01
End Date
2013-09-30
Status
Closed
Released Data Link
Team
Principal Investigator