Methods to study PIDDosome and caspase-2 signalling

The PIDDosome multiprotein complex is formed by PIDD1 and RAIDD (alias CRADD) and serves as activation platform for caspase-2. One of the best characterized triggers for PIDDosome activation is the presence of extra centrosomes, which are frequently observed in cancer and in naturally polyploid tissues such as liver and heart. Depending on the cell type, activated caspase-2 can (1) cleave MDM2, thereby triggering a p53 response, or (2) cleave the BH3-only protein BID to induce apoptosis. Here, we describe our biochemical and cell biological tools to study the PIDDosome and caspase-2 activity. These include methods to experimentally induce PIDDosome formation in cells, as well as techniques to monitor pathway activity via protein and mRNA expression analysis, and flow cytometry. Moreover, we describe how to follow centrosome maturation for PIDDosome activation using immunofluorescence microscopy.