Involvement of the nuclear pore complexes in coordinating entry, progression and exit from mitosis – Nups&cycle

In eukaryotes, the nuclear envelope (NE) separates nuclear and cytoplasmic cellular activities. During interphase, transport of macromolecules between these two compartments occurs through the nuclear pore complexes (NPCs) that are composed of multiple copies of ~30 different proteins termed nucleoporins (Nups). Only a few Nups are integral membrane proteins, and most of the others associate into subcomplexes that ultimately assemble to build up the mature NPCs. Upon cell division in metazoans, characterized by a so-called "open mitosis", the establishment of the mitotic spindle apparatus implies the disassembly of the NE and NPCs, a process termed nuclear envelope breakdown (NEBD). Key structures involved in chromosome congression and segregation are kinetochores, which assemble on centromeres during mitosis. Kinetochores are involved in microtubule-chromosome connection and in the establishment of the mitotic checkpoint. Following chromosome segregation, NE and NPCs reassembly is initiated in late anaphase, and further proceeds until late telophase leading to the reformation of two daughter nuclei. Cell division thus requires a tight spatial and temporal coordination of various events and failure in one of these steps can induce severe defects leading in most cases to either cell death or tumorigenesis. It is now well established that temporal coordination in mitosis is set by the activation or inactivation of a series of regulators, including mitotic kinases and phosphatases. However the completion of each event might also provide a signal for the beginning of the next one, ensuring the correct sequence of mitotic events. In this respect, it is noteworthy that several checkpoint proteins are localized at NPCs in interphase and that at least one kinetochore constituent involved in chromosome alignment, CENP-F, localize at the NE at the G2/M transition. Key players that likely also contribute to the spatio-temporal coordination of mitosis are the small GTPase Ran and the nuclear transport receptors (karyopherins). Indeed, in addition to their requirement for nucleocytoplasmic transport in interphase, these factors play a critical role in NPC reassembly in ana-telophase as well as in mitotic spindle assembly and kinetochore function. In particular, the karyopherin Crm1 is localized at kinetochores where it provides an anchoring site for RanGAP1 and RanBP2/Nup358, two proteins that are localized at NPCs in interphase and involved in kinetochore-microtubule interactions in mitosis. Together with the Ran pathway, the Nup107-160 NPC sub-complex contributes to various aspects of cell cycle progression. Indeed, in vivo and in vitro approaches revealed that it plays a crucial role at a very early stage of NPC assembly. Furthermore, a fraction of the Nup107-160 complex can be found at kinetochores in mitosis. Our recent studies in Hela cells further revealed that this complex contributes to proper kinetochore functions, partly by recruiting Crm1 and RanGAP1-RanBP2 to kinetochores. Finally, the C.elegans Mel-28 nucleoporin and its vertebrate orthologue, Elys, were recently shown to copurify with the Nup107-160 complex, to be required for NE and NPC assembly and to localize to kinetochores in mitosis. How the dual localization and function of a specific subset of proteins at NPCs and kinetochores could contribute to coordinate entry, progression and exit from mitosis remains an open question. To answer to this challenging question, we will (i) further characterize the molecular network underlying the function of the Nup107-160 complex and Elys at kinetochores, (ii) determine how the phosphorylation of these nucleoporins may contribute to the coordination of various mitotic events, (iii) study the implication in various aspects of NEBD of other mitotic regulators sharing a dual localization at NPCs and kinetochores and (iv) identify and characterize novel NPC-associated proteins, physically or functionally linked to the Nup107-160 complex, that may contribute to the coordination of NE and NPC dynamics with entry, progression and exit from mitosis. To better evaluate the relevance of our findings, functional characterization of these nucleoporins and their partners will be performed in both human cells and C. elegans, two model systems that are well established in the laboratories of partner 1 and 2 respectively. Indeed, these two organisms share most components despite obvious differences in kinetochore organization along the chromosomes. By focusing our researches on the NPCs and kinetochores we expect to define the functional implication of the presence of shared components between these structures. Furthermore, our project should have a broader impact on understanding how, why and to which extend cells recycle structural and regulatory proteins. Beyond improving fundamental knowledge, this study should thus also help to improve strategies for drug target definition.