Constitutive exocytosis (hereafter exocytosis), the uninterrupted transport of secretory vesicles to and subsequent fusion with the plasma membrane, is an essential cellular process for nearly all eukaryotes. Exocytosis is critical for the preservation of plasma membrane homeostasis, cell growth and cell division. In exocytosis multiple copies of the exocyst complex tether each secretory vesicle to the plasma membrane. However, the higher-order structure that coordinates the action of these multiple exocysts remains unexplored. Using Saccharomyces cerevisiae, we integrated 2-color particle tracking, single molecule localization microscopy (SMLM) and cryo-correlative light and electron microscopy (cryo-CLEM) to resolve the native choreography of the multiple exocysts and cellular membranes during exocytosis. The quantitative characterization of fluorescent temporal markers endowed the synergy between SMLM and cryo-CLEM that allowed us to reconstruct the time-resolved architecture of tethering, including its structural dynamics and functional annotation. The exocyst higher-order structure, initially 19 nm in radius, rapidly expands while it pulls the vesicle towards the plasma membrane in a stepwise mechanism comprising three main displacement steps that transit through three metastable states. Ultimately, the exocyst higher-order structure stabilizes, securing the vesicle at ~5 nm from the plasma membrane. Unexpectedly, we found that Sec18 mediates the recycling of exocysts after vesicle fusion, a function that emerges as a central mechanism controlling the flow rate of exocytosis. By resolving the fundamental biophysical principles of tethering we bridged the gap between static isolated structures and the dynamic and multimeric nature of exocytosis.
