Seminar

Live-cell structural biology: towards the mechanics of exocytosis

Oriol Gallego, PhD

Structure-function analyses are fundamental to understand the mode of action of the cellular machinery. However, cellular functions result from the concerted action of complicated systems of protein assemblies and other cellular components, which complexity cannot be reconstituted in a tube. For instance, during exocytosis the secretory vesicles are first tethered to the plasma membrane and subsequently fused with it to release the cargo. Each of these steps is executed by different protein complexes that are somehow coordinated in time and space. Additionally, the activity of each complex requires the interaction with specific biological membranes. Thus, despite in vitro techniques can reconstruct isolated protein complexes up to the atomic scale, models where cellular functions are executed by individual protein assemblies are inadequate and intrinsically limited. PICT technique (“Protein interactions from Imaging of Complexes after Translocation”) provides a unique combination of advantages that allow the detection and the quantitative description of macromolecular interactions in vivo. We have combined PICT with fluorescence localization and computational integration of structural data to determine the 3D structure of protein complexes directly in living cells. Using this approach, we have reconstructed the exocyst, a conserved multisubunit assembly that is responsible to tether secretory vesicles during exocytosis (Picco et al., 2017). The 3D architecture of the exocyst bound to a vesicle allowed us to start building a mechanistic model for exocytosis.