Abstract
Purpose: :
In non-polarized cells, conventional lysosomes have been shown to fuse with the plasma membrane in response to a brief increase in intracellular calcium. Lysosome exocytosis provides additional material for plasma membrane repair and may also be a mechanism to get rid of cellular debris. Here, we investigated the polarity and molecular machinery involved in calcium-induced lysosome exocytosis in model epithelia (e.g., Madin-Darby canine kidney (MDCK) cells) and in polarized ARPE-19 cells.
Methods: :
We used the calcium ionophore ionomycin (5-10 µM, 10 min at 37°C) to induce lysosome exocytosis in polarized ARPE-19 and MDCK cells cultured on Transwell filters. Fusion of lysosomes with the plasma membrane was assessed by the appearance of the lysosome-associated membrane protein Lamp-2 on the plasma membrane by confocal microscopy. We also measured the release of the lysosomal hydrolase beta-hexosaminidase into the apical and basolateral medium by a fluorimetric assay. To investigate the role of the cytoskeleton in this process, we used cytochalasin D and nocodazole to depolymerize actin and microtubules, respectively.
Results: :
ARPE-19 and MDCK cells showed an ionomycin concentration-dependent increase in lysosomal exocytosis. After a 10 min treatment with 10 µM ionomycin, ~3-4% of total cellular beta-hexosaminidase was released, predominantly at the basolateral surface, in both cell types. Actin depolymerization increased lysosome exocytosis in agreement with previous reports that cortical actin acts as a barrier to fusion. Nocodazole treatment did not affect exocytosis suggesting that long-range intracellular transport was not involved in lysosome exocytosis.
Conclusions: :
Lysosomes in RPE cells are the site of lipofuscin accumulation, which contributes to the pathogenesis of age-related macular degeneration. Our results show that RPE lysosomes can be induced to fuse with the basolateral membrane and release their luminal contents. Regulated exocytosis of lysosomes may be a viable strategy to help RPE cells decrease their lipofuscin burden.
Keywords: retinal pigment epithelium • cell membrane/membrane specializations • ipofuscin