Plasticity of the nuclear pore complex – NPC-PLASTIC

This project has been performed in yeast, a simple organism but in which these basic functions are conserved and which is easily manipulated genetically, and safely. We use experimental approaches that combined genetics, biochemistry (or chemistry of life), molecular biology and as well as microscopy approaches that allow us to track molecules in living cells. In particular, the present interdisciplinary project proposes to use high-resolution methods and innovative bioimage informatics approaches.

Our recent studies suggested that ubiquitylation and other similar chemical protein modifications could play an important role in the regulation of NPC functions and dynamics. Ubiquitin is a small molecule which, by binding specifically to target proteins dictates the functions of these proteins. In the present project, we have been able to dissect the diversity of such modifications at the nuclear face of the NPC and their dynamics along the cell division. We could demonstrate their involvement in the association with the core NPC with important consequences on the maintenance of genome integrity, but also as their role as sensors of environmental stresses.

The results obtained during this project provide important insights into the role of posttranslational modifications in the dynamics and functions of the nuclear pore, which are now targeted by new therapeutic drugs.

This project led to 4 publications, including 3 with both partner teams as authors, in peer-reviewed international journals which are famous in their field. This project also allowed the development of a free soft for 3D localization that has been adapted as a Plug-In on Fiji/ImageJ and technology transfert of the «Live-SR« system to the GATACA company..

Nuclear Pore complexes (NPC) constitute a unique gate between the nucleus and the cytoplasm that regulates the selective and massive transport of macromolecules between these cellular compartments. Over the past years, the NPC emerged as a “hub” coordinating nuclear transport, gene expression, chromatin organization and genome integrity. However the limited number of NPC per cell imposes a certain "hierarchy" of these different functions in time and space. To dissect at a molecular level how the NPC integrates these functional constraints represents the next challenge in the biological understanding of this fascinating cellular machine.

Extensive research shed light into the molecular architecture and roles of the NPC. But whether dynamic nucleoporin associations, posttranslational or conformational changes, or temporal changes in expression might represent non-exclusive layers of complexity in NPC structure and function has been so far poorly explored. A systematic analysis of the NPC modification by ubiquitin, that we recently performed, led us to the working hypothesis that dynamic post-translational modifications (PTM), and in particular ubiquitin and ubiquitin-like modifications, would control the plasticity of the NPC structural organization and its role as a platform promoting and orchestrating gene expression.

In the present project, we propose to focus on the nuclear side of the NPC-the nuclear basket- and precisely dissect how the “jeu de rôle” of post-translational modifications can control the structure/composition of the NPC during the cell cycle as well as its function in the gene expression program at a single cell level. We will concentrate our efforts on three main objectives. The first one concerns the precise analysis of the PTM -ubiquitylation, sumoylation and eventually phosphorylation-, of the five proteins (or Nups) composing the nuclear basket, their interplay and roles in controlling the dynamics of the NPC. This task should allow us to precisely analyze and dissect the mechanisms responsible for the architectural dynamics of the nuclear basket. Our second objective is to determine the role of PTM of the nuclear basket on the cell cycle progression and NPC inheritance. To analyze the dynamics of Nups with an accurate spatial and temporal resolution, we will use an imaging workflow that we recently developed,and allows for a unique localization precision with appropriate timescale for live yeast cells. The goal of the third task is to understand how the nuclear basket PTMs influences gene expression by controlling both gene gating and expression as well as the spatiotemporal coordinated cascade leading mRNPs from their site of transcription to their site of nuclear exit. For this purpose, we'll combine genomic analyses with our recent methodological development providing a functional imaging of mRNPs formation at high space-time resolution in live yeast at the single cell level.

Approaching such a complexity in the plasticity of the NPC both in time and space requires a combination of experimental approaches allowing a precise dissection of molecular events at a single living cell and NPC level. The yeast model offers the advantage of easy genetic manipulation, strict control of transcription (and in particular of inducible genes), control of expression levels and biochemical knowledge of the PTM machineries. The present interdisciplinary project proposes to combine genetic engineering and biochemical analysis (Partner 1) with the development of innovative high space-time resolution microscopy methods and bioimage informatics approaches (Partner 2). Both partners involved in this project display fully complementary expertise, already used or developed a combination of multiscale approaches and obtained preliminary data that will guarantee the success of this proposal.