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A. Lakkaraju, F. Diaz, R. Schreiner, E. J. Rodriguez-Boulan; Clathrin Adaptors and SNARE Proteins Direct Lysosome Exocytosis in Polarized Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):490.
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To identify the molecular machinery involved in the polarized exocytosis of lysosomes in epithelial cells. Lysosomes can be induced to fuse with the plasma membrane in response to a localized increase in intracellular calcium. This phenomenon is essential for generating an immune response, for repairing torn plasma membranes and can also be exploited as a mechanism to remove cellular debris.
Lysosome exocytosis in response to a calcium ionophore (ionomycin, 5-10 µM, 10 min, 37°C) was studied in polarized filter-grown ARPE-19 cells and in model epithelia, Madin-Darby canine kidney (MDCK) cells. Polarity of lysosome fusion was assessed by (a) the appearance of the lysosome-associated membrane protein Lamp-2 on the cell surface by confocal microscopy and (b) the release of lysosomal hydrolases into the apical or basal compartments by a fluorimetric assay. We investigated the involvement of actin, microtubules and cholesterol by treating cells with pharmacological agents. Roles of the clathrin adaptor AP-1B and the t-SNARE syntaxin 4 in lysosome exocytosis were studied by microscopic and biochemical analyses of stable cell lines either lacking the µ1B subunit of the AP-1 complex and/or exogenously expressing myc-tagged syntaxin 4.
In MDCK cells, lysosomes predominantly fused with the basolateral membrane after ionomycin treatment. Actin depolymerization and cholesterol depletion both induced exocytosis at the apical surface, whereas cholesterol overload inhibited lysosome fusion. In µ1B-knockdown cells, lysosomes fused mainly with the apical membrane. The polarity of syntaxin 4 correlated with the polarity of lysosome exocytosis - basolateral in wild-type cells and non-polar in µ1B-knockdown cells. RPE cells do not express µ1B and exhibit a non-polar distribution of syntaxin 4. In keeping with this, although lysosomes in ARPE-19 cells mainly fused with the basolateral membrane, there was a significant proportion (~40-45%) that fused with the apical surface.
Lysosomes in RPE cells are the sites of lipofuscin accumulation, which contributes to age-related macular degeneration. Our results show that clathrin adaptors, t-SNAREs, membrane cholesterol content and the actin cytoskeleton specify the polarity and extent of lysosome fusion. Identification of the molecular pathway of lysosome exocytosis will help to assess whether this can be a viable strategy to help RPE cells decrease their lipofuscin burden.
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