Journal
BIO-PROTOCOL
Volume 11, Issue 6, Pages -Publisher
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.3958
Keywords
Protein trafficking; RUSH; Cargo sorting; Confocal microscopy; Vesicle trafficking
Categories
Funding
- Deutscher Akademischer Austauschdienst (DAAD)
- Deutsche Forschungsgemeinschaft (DFG)
- National Institutes of Health (NIH)
- Perspective Program (Boehringer Ingelheim Fonds)
- Max Planck Institute of Biochemistry
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The protocol describes a method to use the RUSH system for live-cell microscopy and quantitative analysis of cargo vesicles. The trafficking of protein cargoes can be evaluated by recording videos in 1-min time frames for 45 min, enabling the study of protein trafficking pathways in cells with more detail.
More than 30% of the total amount of proteins synthesized in mammalian cells follow the secretory pathway in order to mature and be properly sorted to their final destinations. Among several methodologies that describe live-cell monitoring of vesicles, the Retention Using Selective Hooks (RUSH) system is a powerful one that allows to visualize cargo trafficking under physiological conditions. The present protocol describes a method to use the RUSH system in live-cell microscopy and a subsequent quantitative analysis of cargo vesicles to dissect protein trafficking. In brief, HeLa cells are transiently transfected with an MMP2-RUSH construct and vesicle trafficking is evaluated by wide-field microscopy, recording videos in 1-min time frames for 45 min. We also present a quantitative approach that can be used to identify kinetics of uncharacterized protein cargo, as well as to evaluate with more detail processes such as ER-to-Golgi vesicle trafficking. [GRAPHICS]
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