CyPOS; a novel pH-sensitive conjugate for investigation of lysosomal physiology in retinal pigment epithelial (RPE) cells

Purpose: lysosomes play a key role in cellular homeostasis by degrading and recycling waste. In retinal pigment epithelial (RPE) cells, lysosomes breakdown shed photoreceptor outer segments (POS). Optimal proteolysis occurs in highly acidified lysosomes (pH ~4-5), the impairment of which leads to incomplete POS degradation and the accumulation of lipofuscin; a contributory factor to RPE atrophy in age-related macular degeneration. Here, we present the novel fluorescent conjugate CypHer5E-POS (CyPOS), which provides accurate, real-time readouts of intraluminal lysosomal pH and cargo processing in RPE cells.<br/><br/>Methods: cypHer5E mono NHS ester was conjugated to isolated POS (CyPOS) and fed to differentiated RPE (ARPE-19) cells (4μg/cm2). Co-localisation between CyPOS and lysosomes (LysoTracker Green and LAMP2) was assessed by the Costes and object-based methods in living and fixed cells 18 hours post exposure. The long-term maintenance of CyPOS in RPE lysosomes was assessed at four timepoints: 18, 48, 72 and 96 hours. Validation of the CyPOS conjugation was performed by rhodopsin labelling. The pH specificity of CypHer5E and CyPOS was investigated via fluorescence readouts following incubation in varying pH buffers (pH 3.5-7.5, 0.5 increments). Lysosome volume with and without CyPOS was calculated via a novel Nyquist correction method. All data arises from 3 independent experiments.<br/><br/>Results: CyPOS robustly co-localised to lysosomes in living (&gt;80%, 97 lysosomes) and fixed (74.4%, 3 fields of view per repeat) RPE after 18 hours, with the remainder either degraded or in other vesicles. CyPOS persisted within lysosomes for up to 96 hours (p=0.037, ANOVA with Dunnett T3 test, 48 vs 96 hours object-based method). The mean/median CyPOS fluorescence readouts were maximal between pH 3.5-5, which corresponds to the intraluminal acidity of functional lysosomes. Furthermore, CyPOS positive lysosomes were 1.41-fold larger than lysosomes without CyPOS cargos (p=&lt;0.0001, two-tailed Mann-Whitney test).<br/><br/>Conclusions: we show evidence of the novel CyPOS probe co-localising with live and fixed RPE lysosomes, where it can be used for long term investigation of lysosomal physiology. We therefore propose CyPOS as a powerful tool to study lysosomal dysfunction in retinopathies.<br/><br/>This abstract was presented at the 2025 ARVO Annual Meeting, held in Salt Lake City, Utah, May 4-8, 2025.