A fluorescence resonance energy transfer-based approach for investigating late endosome–lysosome retrograde fusion events

Abstract

Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome–lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders—Niemann–Pick type C, mucolipidosis type IV, and Sandhoff’s disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.

Mechanisms of Amine Accumulation in, and Egress from, Lysosomes

“…Data from previously analyzed fluorescence resonance energy transfer (FRET)-based, late endosome–lysosome fusion assays for quantitation of hybrid organelle formation rates were utilized for this purpose [23]. Data from these experiments were used to obtain rate constants for late endosome to hybrid organelle trafficking as well as lysosome to hybrid organelle trafficking.…”

“…We hypothesized that amine-induced vacuoles are merely greatly expanded hybrid organelles, which is consistent with our immunofluorescence evaluations. We have recently developed a FRET-based assay that allows us to quantitatively measure the retrograde fusion of lysosomes with late endosomes [23]. Using this assay, we have shown that the lysosomal membrane protein NPC1 is required for efficient hybrid organelle formation (mutations in NPC1 lead to 95% of cases of Niemann–Pick type-C disease , a fatal neurodegenerative lysosomal-storage disorder) [22,23].…”

Niemann–Pick type C disease: cellular pathology and pharmacotherapy

“…In addition to the failure to export cholesterol from the LEL a variety of experiments have detected multiple transport defects in Niemann-Pick cells (Neufeld et al 1999;Liscum 2000;Choudhury et al 2002;Choudhury et al 2004;Pipalia et al 2007;Kaufmann et al 2009;Tharkeshwar et al 2017) including a slowing of the entire endocytic process (Choudhury et al 2004;Tharkeshwar et al 2017). How can an accumulation of a single metabolite have such wide-ranging effects?…”

Heterogeneous but not random: Cargo degradation in phagosomes is kinetically coupled to their intracellular mobility

“…Because this crosstalk was constant during imaging, it was measured and subtracted from the acceptor emission signal. Following previous protocol (Kaufmann, Goldman, & Krise, 2009), the FRET ratio was calculated as the ratio of the emission intensity of acceptor after crosstalk subtraction $55+&167 to the emission of intensity of the donor 86#67 at the donor excitation (561 nm). In the FRET-ratio-vs-time plot, the slope of the increment portion of the FRET ratio curve, named FRET rate, was used for the quantification of phagosomelysosome fusion rate (Figure 2-figure supplement 2).…”