Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Live-cell Vesicle Tracking in the Secretory Pathway Using HeLa Cells.

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.

Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells.

Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling, regulation and trafficking dynamics at the secretory pathway.

Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs.

Matrix metalloproteinases (MMPs) degrade several ECM components and are crucial modulators of cell invasion and tissue organization. Although much has been reported about their function in remodeling ECM in health and disease, their trafficking across the Golgi apparatus remains poorly understood. Here we report that the cis-Golgi protein nucleobindin-1 (NUCB1) is critical for MMP2 and MT1-MMP trafficking along the Golgi apparatus.