RUSH: a system to monitor the transport of newly synthesized α-granule cargo in real time. (A-B) The RUSH system has 2 components: an ER-hook comprising the streptavidin protein fused to the ER retention signal KDEL, and a cargo-reporter composed of a cargo protein (PDGF) fused to streptavidin-binding peptide (SBP) and a fluorescent protein (Cherry). The cargo-reporter is retained in the ER by the ER-hook, which is bound to the KDEL-receptor. Biotin added to the culture media competes for binding to streptavidin, thus releasing the cargo-reporter from the ER-hook and allowing synchronized traffic to the α-granule. The ER-hook and cargo-reporter are cloned in a bicistronic plasmid to ensure all cells expressing the cargo-reporter have it hooked in the ER in the absence of biotin. (C) Confocal fluorescence microscopy analysis of live MEG01 cells cotransfected with the RUSH bicistronic plasmid and markers of different subcellular compartments: ER, blue fluorescent protein 2–KDEL (BFP2-KDEL); Golgi complex, trans-Golgi network protein–GFP (TGNP-GFP). The α-granules were labeled with endocytosed and chased fibrinogen–Alexa Fluor 647 (Fibrinogen-A647). Four-color images were acquired immediately before adding biotin (time = 0 minutes) and 25 and 150 minutes after the addition of biotin.