NPCquant · Investigation of human nucleoporins stoichiometry and intracellular distribution by quantitative mass spectrometry

Investigation of human nucleoporins stoichiometry and intracellular distribution by quantitative mass spectrometry

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. The main goal of the NPCquant project was to determine the stoichiometry of the NPC in human cells using proteomic technologies. During this project: (i) I developed targeted proteomics assays that allow the quantification of all the 32 nucleoporins (Nups) with high accuracy and reproducibility; (ii) I optimized procedures for the isolation of intact nuclei and nuclear envelope from several tissue culture cells that enabled the measurement of the stoichiometry of the fully assembled NPC in situ as well as the determination of its structure by cryo-electron microscopy (cryo-EM); (iii) I compared the NPC composition in several cancer cell lines; (iv) I generated and characterized several stable cell lines expressing inducible microRNAs targeting different Nups. These cell lines provide valuable tools for structural/functional investigation of single NPC components. I revealed the stoichiometry of the 32 components of the human NPC by absolute protein quantification and corroborate my findings using super-resolution microscopy. My data served as a foundation for an integrated structural approach that ultimately revealed the structure of the NPC scaffold, thus tackling a long-standing goal in structural biology. In addition, I was able to show that NPC composition varies between different cancer cell lines via stoichiometric changes of its peripheral components. Other ongoing projects are exploiting the tools that I developed (targeted proteomics assays and cell lines) to elucidate the structure of other NPC sub-complexes and characterize the functional role of individual Nups in nuclear/cytoplasmic transport. The information provided by this project will have a high impact towards the structural elucidation of the human NPC and, consequently, it will help clarifying the mechanisms of nuclear/cytoplasmic transport, in particular the interaction of transport factors with nuclear pores. Therefore, the research proposed has the potential to clarify the involvement of Nups and the nuclear transport machinery in physiological and pathological processes such as cell differentiation and cancer. Moreover, the developed method will be an important tool available for the scientific community to pursue further structural and functional investigations on the NPC.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз