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.
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We acknowledge members of the cell imaging platform of the Institut Curie (PICT-IBISA platform), especially V. Fraisier and P. Paul-Gilloteaux, and members of the Recombinant Antibodies and Proteins Platform, Curie Institute, for their support; M. Romao for technical help with electron microscopy; and E. del Nery for help with development of automated screening. We thank A. Akhmanova (Erasmus Medical Center, Rotterdam), V. Malhotra (Center for Genomic Regulation, Barcelona), A. El Marjou, P. Benaroch, S. Dufour (Institut Curie) for providing us plasmids coding for STIM1-NN, Ii, SBP, VSVGwt and E-cadherin, respectively; and D. Stephens (University of Bristol) for antibody to Sec24; and B. Goud and T. Jones for critically reading the manuscript. This work was supported by the Centre National de la Recherche Scientifique, the Institut Curie, the Agence Nationale de la Recherche (ANR 09-BLAN-0290) and the Incentive and Cooperative Programs from the Institut Curie. V.M. was supported by the European Molecular Biology Organization.
F.P. is the author of a patent application that covers the commercial use of the RUSH system (EP2010/058229 and WO/2010/142785).
Supplementary Figures 1–8 (PDF 13060 kb)
Real-time imaging of the synchronized trafficking of ManII-SBP-EGFP (corresponds to Fig. 3a). HeLa cells were transfected to express Ii-streptavidin as a hook and ManII-SBP-EGFP as a reporter. After 20 h of expression, at time 00:00, release of the reporter was induced by addition of biotin and monitored using a spinning disk confocal microscope. (MOV 5962 kb)
Real-time imaging of the synchronized trafficking of TNFα-SBP-EGFP (corresponds to Fig. 3b). HeLa cells were transfected to express Ii-streptavidin as a hook and TNFα-SBP-EGFP as a reporter. After 20 h of expression, at time 00:00, release of the reporter was induced by addition of biotin and monitored using a spinning disk confocal microscope. (MOV 3462 kb)
Real-time imaging of the synchronized trafficking of SBP-EGFP-E-cadherin (corresponds to Fig. 3c). HeLa cells were transfected to express Ii-streptavidin as a hook and SBP-EGFP–E-cadherin as a reporter. After 20 h of expression, at time 00:00, release of the reporter was induced by addition of biotin and monitored using a spinning disk confocal microscope. (MOV 6718 kb)
Real-time imaging of the synchronized trafficking of VSVGwt-SBP-EGFP. HeLa cells were transfected to express Ii-streptavidin as a cytoplasmic hook and VSVGwt-SBP-EGFP as a reporter. After 20 h of expression, at time 00:00, release of the reporter was induced by addition of biotin and monitored using a spinning disk confocal microscope. (MOV 901 kb)
Real-time TIRF microscopy of TNFα-SBP-EGFP exocytosis (corresponds to Supplementary Fig. 7). HeLa cells were transfected to express Ii-streptavidin as a hook and TNFα-SBP-EGFP as a reporter. Twenty-eight minutes after the addition of biotin, exocytosis at the plasma membrane was observed using TIRF microscopy. Many bursts of exocytosis can be observed in the two cells present in the field. Duration of the acquisition is indicated in min:s.ms. (MOV 1220 kb)
Dual-color, real-time imaging of the synchronized trafficking of TNFα-SBP-EGFP and ManII-SBP-mCherry (corresponds to Fig. 4a–g). HeLa cells were transfected to express Ii-streptavidin as a hook and TNFα-SBP-EGFP (green) and ManII-SBP-mCherry (red) as reporters. Twenty hours after transfection, at time 00:00, the release was induced by addition of biotin. Images were acquired using a confocal spinning disk microscope. (MOV 4468 kb)
Dual-color imaging of the synchronized trafficking of TNFα-SBP-EGFP and SBP-mCherry–E-cadherin (corresponds to Fig. 4h–j). HeLa cells were transfected to express Ii-streptavidin as a hook, TNFα-SBP-EGFP (green) and SBP-mCherry–E-cadherin (red). After 20 h of expression, at time 00:00, release of the fluorescent reporters was induced by addition of biotin and monitored using a spinning disk confocal microscope. Time is indicated in min:s. (MOV 5433 kb)
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Boncompain, G., Divoux, S., Gareil, N. et al. Synchronization of secretory protein traffic in populations of cells. Nat Methods 9, 493–498 (2012). https://doi.org/10.1038/nmeth.1928
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