Synchronization of secretory cargos trafficking in populations of cells

In mammalian cells, secretory proteins are transported to their destination compartment via the secretory pathway. Cargos start their journey in the endoplasmic reticulum and then reach the Golgi complex where they are processed and sorted to be delivered to their target intracellular compartment. To analyze and visualize this flow of proteins, one needs to synchronize their transport. We recently developed the retention using selective hooks (RUSH) system enabling simultaneous and synchronous release of secretory cargos from the endoplasmic reticulum in a population of cells. Here, we describe how to subclone the gene coding for a cargo of interest into a RUSH plasmid and to monitor its synchronized transport along the secretory pathway in fixed samples and in living cells.