Synchronization of secretory protein traffic in populations of cells

• Published in: Nature methods Vol. 9; no. 5; pp. 493 - 498
• Main Authors: Boncompain, Gaelle, Divoux, Severine, Gareil, Nelly, de Forges, Helene, Lescure, Aurianne, Latreche, Lynda, Mercanti, Valentina, Jollivet, Florence, Raposo, Graça, Perez, Franck
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.05.2012; Nature Publishing Group
• Subjects: 631/1647/1407; 631/1647/245; 631/80/2023; Biochemical assays; Bioinformatics; Biological Microscopy; Biological Techniques; Biological Transport; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biotin; Carrier Proteins - metabolism; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cell research; Cellular biology; Cellular proteins; Golgi Apparatus - metabolism; Golgi Apparatus - ultrastructure; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Life Sciences; Methods; Microscope and microscopy; Microscopy, Confocal - methods; Microscopy, Immunoelectron; Observations; Physiology; Proteins; Proteomics; Retention; Scientific imaging; Streptavidin - metabolism; Testing; Transfection - methods; Vitamins; France
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.1928

The biotin-reversible interaction between a 'hook' protein localized to a particular cellular compartment and a reporter protein of interest is exploited in a simple system to synchronize protein traffic through the secretory pathway. 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.