An increasing number of distinct functions have been assigned to different types of ubiquitin modifications (mono ubiquitin versus ubiquitin chains). In these processes Ub acts as a signalling component able to trigger molecular events in cells. We have shown that the UBAN domain of NF-КB essential modulator (NEMO) binds specifically to linear Ub chains and that this step is both required and sufficient for NF-КB activation. More recently, we have identified novel components of the linear ubiquitin assembly complex (LUBAC) that catalyse linear ubiquitination of NEMO and control the NF-КB and cell death apoptotic and necroptotic pathways downstream of TNF receptors. Using the different structural and biochemical properties of different ubiquitin chain linkages, we have developed fluorescence-based sensors to monitor real-time localization and dynamics of ubiquitination events in vivo. For example, linear chain-specific sensors labeled the ubiquitin coat surrounding cytosolic Salmonella and were also able to block TNF-induced NF-КB activation. New data about the role of LUBAC ligase in the formation of linear chains and physiological regulation of the NF-kappaB pathway and autophagy will be presented.