Abstract
Purpose :
The retinal pigment epithelium (RPE) has a high natural requirement for lysosomal degradation due to the daily phagocytosis and clearance of photoreceptor outer segments. Lysosomes are known to be heterogeneous in terms of morphology and content of classical lysosomal markers, but the functional significance of this is unclear. We aimed to characterise lysosomal heterogeneity in the RPE in order to determine the extent to which different lysosomal subpopulations represent stages in the lysosomal life cycle, which maintains the degradative capacity of the cell.
Methods :
Morphology and content of lysosomal markers in primary porcine RPE and other cell models was determined by conventional and immuno-electron microscopy. To determine accessibility of lysosomes to the endocytic pathway cells were loaded with either fluorescent dextrans or colloidal gold particles and examined by live cell confocal microscopy and electron microscopy. Electron tomography was used to assess the 3-dimensional structure of RPE lysosomes.
Results :
Vacuoles positive for lysosomal membrane proteins could be subdivided into those composed exclusively of lamellae, those with lamellar regions and those completely lacking lamellae. Endocytosed BSA gold particles were found exclusively in non-lamellar regions whilst the lamellar regions contained much of the lysosomal acid hydrolase, cathepsin D. Pulse chase experiments employing BSA gold showed that after degradation of the BSA that stabilises the gold particles, undigested aggregated gold particles were segregated into non-lamellar lysosomes. Parallel experiments employing sequential pulses of dextrans labelled with different fluorophores, separating the two pulses by up to 7 days, showed that a proportion of dextran-containing lysosomes lose accessibility to the endocytic pathway, suggesting that they have dropped out of the lysosome cycle. Electron tomography indicated multiple connections between lamellar lysosomes whilst at least some non-lamellar lysosomes have lost connection with this lysosomal network.
Conclusions :
Morphologically heterogeneous lamellar and non-lamellar lysosomes within the RPE represent different stages in the lysosome cycle. Undigestible material is segregated into non-lamellar lysosomes that appear to lose the ability to interact with endosomes and lysosomes and drop out of the lysosome cycle.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.