ARTICLES NATURE METHODS | VOL.9 NO.5 | MAY 2012 | 493 To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post- Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport. It is now clear that there are many routes for proper transport, modification and addressing of proteins in the secretory pathway of cells. This had been anticipated because of the diversity of target compartments (for example, plasma membrane, endosomes and lysosomes) but also because of the existence of plasma membrane subdomains 1–3 . Even for a simple transport route (such as the endoplasmic reticulum (ER) to plasma membrane) in nonpolar- ized cells, several independent pathways support the traffic of different subsets of protein cargo 4–7 . A comprehensive view of the mechanisms and dynamics of cargo sorting in these multiple secretory pathways requires assays that allow the dissection of the routes specific cargos follow. Ideally, these assays should be adaptable to a large diversity of cargos, allow quantitative and real-time trafficking observations and be amenable to large-scale experiments. Such assays would enable more detailed study of the large number of diverse trafficking regulators, such as families of small GTPases or SNAP receptors, the majority of which remain functionally unannotated. Several approaches are currently used to image secretory traffic. For instance, short-term reporter expression, either after DNA microinjection or after photoconversion or photobleaching of a Synchronization of secretory protein traffic in populations of cells Gaelle Boncompain 1,2 , Severine Divoux 1,2 , Nelly Gareil 1,2 , Helene de Forges 1,2 , Aurianne Lescure 3 , Lynda Latreche 3 , Valentina Mercanti 1,2 , Florence Jollivet 1,2 , Graça Raposo 1,2 & Franck Perez 1–3 fraction of the target protein in single cells, has been used for this purpose. These methods are frequently coupled with temperature block and release to synchronize the reporter pool and allow traffic through the secretory pathway to be followed quantitatively. Temperature block (for example, 15 °C to block proteins in the ER or 20 °C to block proteins in the Golgi 8,9 ) has proven a powerful tool for studying intracellular traffic, including in living cells, but it also imposes a temporary arrest of virtually all biosynthetic pathways in a mammalian cell. Another classical and powerful method for cargo synchronization relies on a thermosensitive viral glycoprotein (VSVGtsO45) that cannot exit from the ER at the restrictive temperature of 39.5 °C (refs. 10,11). Synchronous transport and processing at 32 °C can be monitored in living cells 12–14 . However, the restrictive and permissive temperatures for this system are not fully physiological and complicate the use of this system for systematic screening. In addition, the VSVGtsO45 system cannot be easily adapted to the analysis of a large diversity of cargos. An alternative method 15 relies on the fusion of the pro- tein of interest with conditional aggregation domains, resulting in the aggregation of the fusion protein in the ER. This aggrega- tion is reversed by the addition of a small ligand, allowing syn- chronous and controlled secretion of soluble or transmembrane proteins 15–18 . However, this approach is not applicable to proteins that cannot be tagged on their luminal domains and cannot be used for synchronization after the ER. A regulated trafficking system has also been developed that depends on the induction of reporter expression in Drosophila melanogaster cells 19 . It has been used for screening 20,21 but because of slow kinetics would be less adapted to real-time analysis of early trafficking steps (such as ER export). Thus there is still an unmet need for a versatile trafficking assay that allows efficient synchronization of diverse cargo, under physiological conditions, permits quantitative live cell imaging and is amenable to automated screening. Here we describe such a system, named RUSH, which relies on the selective retention and release of cargo molecules from a donor compartment. We showed that the RUSH system can be used to study and quan- tify the trafficking of diverse proteins in live cells or in end-point assays, and that it shows potential for automated quantitative imaging and screening in the future. 1 Institut Curie, Centre de Recherche, Paris, France. 2 Centre National de la Recherche Scientifique, Unité Mixte de Recherche 144, Paris, France. 3 The BioPhenics Laboratory, Institut Curie, Paris, France. Correspondence should be addressed to F.P. (franck.perez@curie.fr). RECEIVED 2 AUGUST 2011; ACCEPTED 24 JANUARY 2012; PUBLISHED ONLINE 11 MARCH 2012; DOI:10.1038/NMETH.1928 npg © 2012 Nature America, Inc. All rights reserved.