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NucEnv Report Summary

Project ID: 322582
Funded under: FP7-IDEAS-ERC
Country: Switzerland

Final Report Summary - NUCENV (Nuclear Envelope Biogenesis, Function and Dynamics)

The nuclear envelope (NE) forms the boundary of the nuclear compartment in eukaryotic cells. It serves as a protective shell of the genome and a versatile communication interface. The architecture of NE is perfectly adapted to these functions. The boundary task is fulfilled by double membrane formed by a specialized membrane sheet of the endoplasmic reticulum (ER) that is highly asymmetric with respect to protein composition. The inner nuclear membrane (INM) is mechanically supported by the intermediate filament network of the nuclear lamina and contains a unique set of membrane proteins that aid chromatin organization and the regulation of gene expression. Nucleo-cytoplasmic communication occurs primarily through large nuclear pore complexes (NPCs) that allow for the selective bidirectional nucleo-cytoplasmic exchange of macromolecules. In addition, Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, composed of SUN and KASH proteins, bridge the NE and provide molecular handles for nuclear migration and anchorage as well as for chromosome movements.
The correct targeting of proteins to the inner nuclear membrane (INM) presents the basis for establishment of NE asymmetry and is important for cellular and organismal homeostasis. Exploiting a newly developed visual kinetic assay for protein targeting to the INM relying on semi-permeabilized mammalian cells, we have derived a mathematical description of the targeting process and established that INM targeting is governed by the principles of diffusion-retention. We have further demonstrated that the efficient targeting of membrane proteins from the ER to the NE requires continuous nucleotide hydrolysis by members of the ER-resident Atlastin GTPase family for the maintenance of ER topology, thereby assuring the efficient diffusion-based distribution of macromolecules within the network – a finding that bears importance for other ER-associated protein sorting events. Collectively, our work suggests a unifying mechanism governing the targeting of proteins to the INM in mammalian cells.

While the NE functions as a selective and rather static boundary of the nuclear compartment during interphase of the cell cycle, the NE is dismantled during open mitosis. We have deciphered two aspects of the molecular mechanisms underlying NE breakdown in mammalian cells. Firstly, our work established that LINC complexes facilitate the microtubule-dependent remodeling of NE membranes at the onset of open mitosis, which is a process accompanying the formation of a cytoplasmic mitotic spindle. Secondly, using an in vitro system that recapitulates mitotic NPC disassembly, we have demonstrated that mitotic NPC disassembly is a phosphorylation-driven process, governed by the concerted action of multiple protein kinases that together promote NE permeabilization and the disassembly of the central NPC scaffold. We showed that multisite protein phosphorylation of the key linker nucleoporin Nup53, a component of the inner ring complex, liberates Nup53 out of its protein-protein interaction network during mitotic entry. Based on our work, phosphorylation-driven unzipping of nucleoporin-nucleoporin interactions emerged as a key mechanism of mitotic NPC disintegration of NPCs. This concept also explains as to how cells can disassemble supramolecular complexes like NPCs on a relatively short time scale during their rapid reorganization for open mitosis.

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