Sublethal photic stress and the motility of RPE phagosomes and melanosomes.

Pigment granules (melanosomes), phagosomes, and other membrane-bound organelles translocate within cells along cytoskeletal scaffolds of microfilaments or microtubules.1-4 Both proteins constituting these scaffolds, actin and tubulin, are sensitive to modification under conditions of oxidative stress, which can produce cytoskeletal disruption.5-11 This observation motivated us to analyze whether oxidative stress impairs organelle movement. Stress-induced impairments of organelle motility, should they occur, could affect the ability of cells to maintain normal subcellular distribution of organelles and thus their overall cytoplasmic organization. A focus of this investigation was the motility of melanosomes within retinal pigment epithelial (RPE) cells subjected to oxidative stress induced by irradiation with visible light. This issue is of particular interest for several reasons. The retinal pigment epithelium is a monolayer of long-lived, postmitotic pigmented cells subjected to a lifetime of visible light irradiation. Photic damage to the retinal pigment epithelium caused by visible light, especially blue light, is believed to cause tissue dysfunction over time, contributing to age-related degenerations of the adjacent photoreceptors that the retinal pigment epithelium supports.12,13 Among the age-related structural changes that take place within RPE cells is redistribution of melanosomes from predominantly apical to more basal regions of the cell cytoplasm,14 suggesting that aging may affect mechanisms used to localize organelles. The functions that melanosomes perform within pigmented cells are not fully understood, but optical screening by melanin pigments is a well-established property of the organelle. Given that light strikes the retinal pigment epithelium from the apical side, the function of screening light-sensitive molecules in the RPE cytoplasm is best performed when the pigment granules are located apically. Melanin is also believed to protect cells from oxidative stress by virtue of its antioxidant properties.15 Little is known about the subcellular location of pro-oxidant and antioxidant species within cells, but the positioning of melanosomes within pigmented cells may be one determinant of whether the granules are located near sites where reactive oxygen species are generated so that the pigment can exert its putative antioxidant effect. To determine whether organelle motility in RPE cells is susceptible to oxidative stress induced by light, isolated porcine melanosomes were introduced by phagocytosis into cultures of the human RPE cell line ARPE-19. Parameters were then established for subjecting cultures to sublethal stress using visible light irradiation. We focused on inducing low levels of stress that might produce small functional decrements rather than overt cell death. Mild stress is likely more relevant during aging, when stress is not acute and fatal but rather subacute and chronic or recurrent and when it produces mild damage that could have cumulative consequences over time. Accordingly, methods were devised to quantify organelle motility in stressed cells that remained viable. Although RPE melanosome movement was one focus of this investigation, in most experiments the pigment granule was phagocytized and, therefore, encapsulated within a phagosome. This model of phagocytized granules has several benefits for analyses of organelle movement and photic stress. Granule uptake can be adjusted to control particle number within cells, and, because the granule is dark, phagosomes containing them can be tracked by bright-field microscopy, thereby avoiding the use of fluorescent tags that could confound studies involving light treatment. Further, as explained later, properties of the pigment can be exploited to alter the magnitude and location of photic stress. In addition to phagocytized melanosomes, a few experiments were conducted to determine the effects of photic stress on other organelles: phagosomes containing black latex beads in ARPE-19 cells and endogenous melanosomes in primary cultures of porcine retinal pigment epithelium.