Use of Protein interactions from Imaging Complexes after Translocation (PICT) to characterisein situthe spatial configuration of proteins interacting with the exocyst

Abstract

ABSTRACTAlthough the structure of the exocyst has been successfully resolved by cryo-electron microscopy, multiple studies showed that exocyst function requires the transient interaction with additional proteins. Unfortunately, the exocyst-interacting network could not be collectively reconstituted, challenging the understanding of how the exocyst complex is coordinated within the network of proteins involved in exocytosis. In a previous work, we described an approach that combines Protein interactions from Imaging Complexes after Translocation (PICT) and centroid localization analysis of diffraction-limited fluorescence signals to estimate the distance between a labelled protein and a spatial reference. This approach allows resolving the spatial organisation of protein interactions directly in living cells, both for intra-complex (i.e. between exocyst subunits) and inter-complex (i.e. between exocyst and transient binding proteins) interactions. In this chapter, we present the protocol to reproduce the sample preparation and image acquisition for PICT experiments. We also describe the computational image analysis pipeline to estimate the distance in PICT experiments. As illustration of the approach, we measure the distance from the spatial reference where the exocyst is anchored to 1) an intra-complex interaction (i.e. Sec5 exocyst subunit) and 2) an inter-complex interaction (Sec2, a guanyl-nucleotide exchange factor mediating vesicle tethering).