Retention Using Selective Hooks-Synchronized Secretion to Measure Local Exocytosis.

Proteins destined to be exposed to the extracellular space enter the secretory pathway at the level of the endoplasmic reticulum. Proteins are then transported to the Golgi apparatus and addressed to their destination compartment, such as the plasma membrane for exocytic cargos. Exocytosis constitutes the last step of the anterograde transport of secretory cargos. Exocytic vesicles fuse with the plasma membrane, releasing soluble proteins to the extracellular milieu and transmembrane proteins to the plasma membrane. In order to monitor local exocytosis of cargos, we describe in this chapter how to perform synchronization of the anterograde transport of an exocytic cargo of interest using the retention using selective hooks (RUSH) assay in combination with selective protein immobilization (SPI). SPI is based on the coating of coverslips with anti-green fluorescent protein (GFP) antibodies, which capture the GFP-tagged RUSH cargos once exposed to the cell surface after its release by the addition of biotin.