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

Background

Monitoring vesicle trafficking is an excellent tool for the evaluation of secretory protein dynamics in living cells. Given that around 30% of the total amount of newly synthesized proteins in mammals follow the secretory pathway (Pfeffer, 2010; Boncompain and Weigel, 2018), the study of its trafficking dynamics is key for the understanding of protein sorting and secretion. Proteins that follow the secretory pathway go through several maturation steps, starting at the ER, they are transported along the different Golgi stacks until they reach the trans-Golgi network (TGN), a sorting station. In the TGN they are finally packed into vesicles and initiate their route to their final destination (Glick and Luini, 2011; Pantazopoulou and Glick, 2019).

Recent developments in microscopy, as well as new methodologies developed to study the synchronized trafficking of proteins allowed to better understand signaling, regulation and trafficking dynamics at the secretory pathway (Stephens and Perez, 2013). One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition (Boncompain et al., 2012).

The RUSH system has two main elements: a protein of interest (POI) tagged with a fluorophore and bound to a streptavidin binding peptide (SBP); and a streptavidin molecule bound to a retention signal (known as the “hook”, e.g., a KDEL sequence for retention in the ER; Figure 1). This complex, in the absence of biotin will be retained in the donor compartment, but once biotin is added to the culture medium, the POI-fluorophore-SBP complex will be released to the next compartment. This set-up allows to follow up the POI at different time points by confocal microscopy (Boncompain et al., 2012).

Figure 1. Scheme representing the RUSH system. A. Protein complex containing the protein of interest (here matrix metalloprotease 2, MMP2), a streptavidin binding peptide (SBP) and a fluorescent protein (eGFP). The complex is bound via SBP to streptavidin (Str), which is linked to a KDEL sequence for its retention in the endoplasmic reticulum (donor compartment). Without biotin addition, the complex remains in the donor compartment; however, once biotin is added to the media, it binds to streptavidin and enables the trafficking of the MMP2-SBP-eGFP protein complex to the acceptor compartment. Figure adapted from Boncompain et al. (2012). B. Immunofluorescence images showing the trafficking of MMP2 across the secretory pathway, with MMP2 localizing at the ER when biotin is absent, and later localizing at the Golgi (15 and 30 min) and in post-Golgi vesicles (45 min) upon biotin addition. TGN46: trans-Golgi marker. Figure taken from Pacheco-Fernandez et al. (2020).

Among other techniques, the RUSH system has the advantage of enabling the monitoring of cells under physiological conditions. Given that biotin is a non-toxic small molecule, the synchronization of cargo release does not lead to high stress for the cells (Boncompain and Perez, 2013). Also, compared to other techniques such as the light-triggered protein secretion system (Chen et al., 2013), it does not require a previous aggregation of the protein, which may lead to trafficking through different routes, higher protein degradation rates, and consequently, higher cell toxicity (Boncompain and Perez, 2013).

Building on the original RUSH methodology, here we present a protocol that allows to quantitatively evaluate protein cargo trafficking at different fixed time points. Importantly, in this protocol we provide a quantitative analysis to better examine intra-Golgi trafficking dynamics by evaluating specific cargo trafficking dynamics along the secretory pathway using confocal microscopy. This allows to evaluate the impact of knocking-out or silencing secretory pathway proteins on cargo trafficking (Crevenna et al., 2016; Deng et al., 2018; Pacheco-Fernandez et al., 2020).