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

Background

More than 30% of the total amount of proteins synthesized in mammalian cells follow the secretory pathway (Pfeffer, 2010; Boncompain and Weigel, 2018). Through this pathway, proteins mature by trafficking from the ER and along the different Golgi stacks until they reach the trans-Golgi network (TGN), where they are finally sorted and packed into vesicles that will be delivered to other organelles within the cell, or to the extracellular milieu (Glick and Luini, 2011; Pantazopoulou and Glick, 2019).

In the recent years, several reports in the literature document advance in the live-cell monitoring of vesicles that follow the secretory pathway (Stephens and Perez, 2013). One of the most helpful tools is the Retention Using Selective Hooks (RUSH) system, a methodology that enables the synchronized monitoring of fluorescent vesicles in a living cell upon biotin addition (Boncompain et al., 2012). In this system, cells are either transiently transfected or stably express a two-part protein complex consisting of a protein of interest (POI) tagged with a fluorophore and bound to a streptavidin binding peptide (SBP), and streptavidin bound to a retention signal (known as the “hook”, e.g., a KDEL sequence for retention in the ER; Figure 1). Given the higher affinity of streptavidin to biotin, once the latter is added to the cell culture media, the SBP-POI complex is released to the next compartment and the trafficking can be followed either live by wide-field microscopy or monitored at different fixed 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) is bound via SBP to streptavidin (Strep), which is linked to a KDEL sequence for the retention of the complex in the donor compartment (in this example, the ER). Once biotin is added to the media, it binds to streptavidin, enabling the MMP2-SBP-eGFP protein complex to travel to the acceptor compartment (the Golgi apparatus in the secretory pathway). Figure taken from Pacheco-Fernandez et al. (2020) and adapted from Boncompain et al. (2012).

One of the main advantages of using the RUSH system is that it enables to carefully dissect the trafficking kinetics of soluble and membrane proteins that follow the secretory pathway under physiological conditions (Boncompain et al., 2012). Some other techniques developed in recent years have also enabled the characterization of vesicle trafficking through the secretory pathway. Among others, super-resolution confocal live imaging (Kurokawa et al., 2019); synchronization of cargo-retention and release by polymerization/depolymerization (Cancino et al., 2013) and a light-triggered protein secretion system (Chen et al., 2013). However, such techniques require either a more complex set up, the use of more sensitive equipment, or the aggregation of the protein of interest previous to its synchronized trafficking, which may lead to cargo trafficking through different pathways than those followed by the unaggregated protein (Boncompain and Perez, 2013).

Based on the original RUSH methodology developed by Boncompain et al. (2012), here we present a protocol that provides in addition a quantitative analysis, which aims to better dissect intra-Golgi trafficking by counting the number of cargo vesicles in 1-minute time video frames during a defined period of time, in order to assess post-Golgi cargo sorting kinetics (Deng et al., 2018). Furthermore, the ImageJ macro developed for such analysis is also optimized for dissecting the ER-to-Golgi cargo trafficking kinetics using Golgi compaction (ratio between Golgi:ER fluorescent signal per minute) as a measure (Pacheco-Fernandez et al., 2020).