Background

Cells are constantly exchanging materials with their extracellular environment and in this process they internalize cargo in vesicles at the plasma membrane. This internalized cargo is delivered to the early endosomes from where it either goes back to the plasma membrane via recycling endosomes or enters the canonical endocytic pathway. Once destined to be degraded, the cargo moves to late endosomes and finally fuses with lysosomes where the active hydrolases digest the cargo (Jovic et al., 2010). These endocytic compartments have characteristic pH of their lumen. The early endosomes have a pH in the range of 5.9-6.8, late-endosomes have pH range of 4.9-6.0, and lysosomes are most acidic with pH ranging from 4.5-5.0 (Maxfield and Yamashiro, 1987). The acidic environment of lysosomes is necessary for the activity of hydrolases present in its lumen and degradation of cargo (Garg et al., 2011; Khatter et al., 2015a and 2015b).

Here we discuss the trafficking assay using DQ-Red BSA as cargo, which is a BSA (bovine serum albumin) derived cargo heavily labeled with BODIPY TR-X dye, resulting in self quenching of the dye. Degradation of DQ-Red BSA in acidic, hydrolase active endo-lysosomes results in smaller protein fragments that have isolated fluorophores, hence de-quenching the dye that can be visualized as a bright fluorescence in cells. The excitation and emission maxima for this dye are ~590 nm and ~620 nm respectively. Trafficking of DQ-Red BSA can be used to study the delivery of cargo to lysosomes (Pols et al., 2013; Marwaha et al., 2017; see Figure 1). Under normal conditions, DQ-Red BSA traffics to lysosomes and is cleaved by lysosomal hydrolases, resulting in bright red fluorescent signal (Figure 2). Disruption of cargo delivery to lysosomes such as by depletion of a certain gene product or treatment with chemical inhibitors (such as Bafilomycin A) impairs proteolysis of DQ-Red BSA and thus weak or no fluorescence is observed (see Figure 3).


Figure 1. Schematic representation of DQ-Red BSA cargo uptake and processing in cells. DQ-Red BSA is endocytosed in cells and traffics through early endosomes to late endosomes which then fuse with acidic hydrolase containing lysosomes. This leads to the formation of endo-lysosomes that degrade DQ-Red BSA, de-quenching the fluorescence of the dye attached to this cargo. DQ-Red BSA is heavily labeled with fluorescent dyes that lead to quenching of their fluorescence (shown by pink fluorophores attached to the cargo). Once DQ-Red BSA has reached the degradative endo-lysosomes, the cargo is broken down into smaller fragments which lead to the de-quenching of the fluorophores (shown as bright red spots in endo-lysosomes).


Figure 2. De-quenching of DQ-Red BSA fluorescence at different time points post endocytosis. Representative single-plane confocal micrographs of HeLa cells showing fluorescence (red signal) of DQ-Red BSA after an uptake of 1 h (A) and 6 h (B) time points. At the end of the uptake time point, cells were fixed and stained with DAPI (blue) to mark the cell nucleus. A bright fluorescence punctae of DQ-Red BSA is visible post 6 h uptake as compared to 1 h, indicating its delivery to acidic compartments of the cell. Scale bars = 10 µm.


Figure 3. DQ-BSA uptake and delivery to lysosomes. Representative single-plane confocal micrographs of HeLa cells incubated with DQ-Red BSA in 1% serum containing media for indicated time point in the absence (A) or presence (B) of 100 nM Bafilomycin A1, a V-ATPase inhibitor that disrupts lysosome fusion. The cell nucleus is stained using DAPI (blue). Scale bars = 10 µm.