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J. M. Burke, M. Zareba, M. M. Henry, T. Sarna; Photobleaching of RPE Melanosomes Affects Protein Oxidation and Susceptibility of Melanosome-Filled RPE Cells to Photic Stress. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5071.
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© ARVO (1962-2015); The Authors (2016-present)
Melanin has antioxidant properties but the melanin content of RPE cells declines with aging, perhaps due to photobleaching, raising the possibility that photobleaching changes the melanosome's antioxidant and cytoprotective functions. Here RPE melanosomes were experimentally photobleached and new assays were developed to analyze effects on granule-associated protein oxidation and on survival of photically stressed RPE.
Porcine RPE melanosomes were photobleached with visible light for intervals to 24 hr. Untreated or photobleached melanosomes, or control black latex particles, were coated with actin (a protein that is sensitive to oxidation and associated with organelle surfaces) and illuminated in a photosensitized (Rose Bengal) cell free system. Protein was stripped from particles, then blotted for actin or protein carbonyls and quantified by densitometry. For cell survival analysis, ARPE-19 cells containing phagocytized particles were illuminated with blue light and analyzed dynamically by live cell imaging to identify the time of apoptotic blebbing in individual cells containing high particle numbers (>30).
Actin associated with untreated melanosomes showed lower carbonylation than actin associated with control particles, but photobleaching markedly increased oxidative protein modification, both of actin and of endogenous melanosomal protein(s). Untreated melanosomes did not protect ARPE-19 cells from photic injury as predicted; cell survival was less than for cells with control particles. However, survival was even worse for cells containing photobleached melanosomes, which showed an earlier onset of blebbing and fewer surviving cells after illumination.
Experimental photobleaching of RPE melanosomes makes cells containing them more sensitive to light-induced cytotoxicity and reduces the particles’ ability to influence oxidative protein modification. How melanosomes (photobleached or not) interact with their local subcellular environment to modify cell survival is poorly understood and is likely determined by both melanin content and surface properties of the organelle.
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