Andreas Marx / Martin Scheffner / Andreas Zumbusch
Visualizing ubiquitin processing in cells
Experimental approaches to study ubiquitylation have been dominated by genetic and biochemical analyses. By contrast, complementing imaging-based approaches for studying ubiquitylation within cells are still in their infancy. The overarching aim of this project is to develop and apply approaches for live cell imaging of different aspects of ubiquitylation. Firstly, we will monitor the activity of the ubiquitin-activating enzyme UBA1 with spatial and temporal resolution in living cells. This requires the synthesis of dedicated nucleotide probes, engineering of UBA1, and advancing the capability of an existing wide-field FLIM microscope to image FRET changes within cells. Secondly, we will develop ubiquitin chain-specific binding proteins and apply these for the identification of respectively modified proteins. The dynamics of the ubiquitylation of these proteins shall be studied in cells in real time using the aforementioned advanced fluorescence microscopy methods.