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Kimberly A Toops, Li Xuan Tan, Aparna Lakkaraju; Complex anomalies in late endosome and lysosome trafficking in stressed retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4666.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) depends on its endo-lysosomal network for degrading and recycling phagocytosed photoreceptor outer segments. With age, declining efficiency of this system leads to the formation of aggregates within (lipofuscin bisretinoids) and around (drusen) the RPE. Bisretinoids, visual cycle byproducts that accumulate in RPE lysosomes, increase lysosomal pH and trap excess cholesterol. Late endosomes and lysosomes are transported along microtubules within the cell. We recently showed that bisretinoids increase tubulin acetylation on stable microtubules, which interferes with autophagosome trafficking and autophagy. Here, we investigated how bisretinoids and cholesterol affect the organization and microtubule-mediated transport of late endosomes and lysosomes.
Adult primary RPE were genetically or chemically labeled with markers for late endosomes, lysosomes, microtubules and cholesterol. Control RPE and cells treated with the bisretinoid A2E were imaged live using high-speed spinning disk confocal microscopy (Andor). 4D image analysis (Imaris) was performed to quantify organelle numbers, morphology and trafficking. RPE lysates from wild type and Stargardt disease (Abca4-/-) mice were immunoblotted for endo-lysosomal proteins.
Analysis of live imaging data showed intriguing differences between late endosomes and lysosomes in the RPE. We observed fewer late endosomes, with limited long-range movements in cells with A2E, compared to control RPE. These data were confirmed in vivo: Abca4-/- RPE had less CD63 (a marker for late endosomes), compared to age-matched wild types. Lysosome numbers were unchanged in A2E-treated cells, but lysosome volume increased. Lysosomes in cells with A2E exhibited trafficking anomalies in the population with displacements > 2 µm: these moved faster with altered microtubule association and frequent directional switches.
Our data indicate that subtle defects in morphology and transport could contribute to a slow loss of endo-lysosomal degradative and signaling functions in RPE with bisretinoids. Importantly, these anomalies are deviations from normal trafficking patterns, as opposed to a total cessation of organelle trafficking. Live imaging can provide valuable insight into organelle traffic and function in the RPE and help identify key vulnerabilities that can promote diseases like macular degenerations.
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