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Investigation of human nucleoporins stoichiometry and intracellular distribution by quantitative mass spectrometry (NPCquant)
Start date: Apr 1, 2012, End date: Mar 31, 2014 PROJECT  FINISHED 

The nuclear pore complex (NPC) is one of the most intricate multi-protein assemblies found in eukaryotic cells encompassing ~30 different components called nucleoporins (Nups). The structural determination of the NPC represents a major challenge due to its size and location in the nuclear envelope. Accurate quantitative data on NPC composition that are required to generate structural models at atomic resolution are currently lacking. Here, I propose to apply targeted mass spectrometry to study the stoichiometry and intracellular distribution of the human NPC components. For this purpose, I will establish an assay based on selected reaction monitoring (SRM) experiments that will allow the absolute quantitation of Nups in different cell compartments. The assay will be used to systematically analyze the effect of Nup perturbation on NPC composition by screening cell lines where single Nups are depleted by gene silencing. The compositional analysis of the gene silencing phenotypes will provide the base for complementary structural investigations performed using cryo-electron tomography. The integration of proteomic and structural data will provide accurate information on the stoichiometry and spatial arrangement of Nups within NPCs. In addition, by mapping the intracellular distribution of Nups, it will be possible to gain insights into their functional roles beyond transport. The multidisciplinary approach that will be developed has the potential to pave the way towards the integration of quantitative proteomic technologies in structural studies of other cellular substructures. This fellowship will give me the opportunity to lead an ambitious project at the interface between proteomics and structural biology, and to work in a collaborative interdisciplinary environment. The scientific and complementary skills I will acquire and the opportunity to work in a leading European institution will be fundamental for my development towards an independent scientific career.
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