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Protein-engineering based approach for super-resolution imaging of nucleoporin-chromatin interactions (NUPTAG)
Start date: Jan 1, 2014, End date: Dec 31, 2015 PROJECT  FINISHED 

Nucleoporins are major building blocks of the nuclear pore complex (NPC). In addition to their role in the nucleocytoplasmic transport, a subset of nucleoporins have additional gene regulatory roles. They can bind chromatin and chromatin modifiers, either directly at the NPC or in the nucleoplasm, leading to gene repression or activation. Due to a lack of techniques that allow mapping spatial and temporal heterogeneities of individual proteins with high precision, the molecular mechanisms underlying these phenomena are far from being understood. To overcome this, I will develop a combined protein-engineering/super-resolution approach for single amino acid targeted labelling and tracking of proteins inside the cells. This strategy is based on the translational incorporation of newly developed non-canonical amino acids (ncAAs). These ncAAs are suitable for inverse-electron-demand Diels-Alder reaction, a special type of highly selective and rapid “click-chemistry” reactions proceeding in a non-toxic and fully biocompatible way inside living cells. I will employ this technology to systematically label a set of nucleoporins and chromatin modifiers with small dyes. The high photostability of these fluorophores permits long-term imaging and will allow me to identify nucleoporin binding partners in cultured mammalian cells with nanometer resolution. This will set the stage for a technology that enables deciphering protein plasticity in vivo at the molecular level and provide novel insights into multifunctionality of nucleoporins.

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