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M. Zareba, A. Herrnreiter, J. M. Burke; Oxidative Stress Induces Loss of Proteins Mediating Organelle Motility From RPE Phagosomes and Impairs Their Movement. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2154.
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Phagosome movement in RPE cells slows when cells are subjected to sub-lethal oxidative stress induced by visible light. Here we analyzed the cytoskeletal scaffold and motor protein complexes that support organelle motility to determine whether motility machinery is stress sensitive.
Isolated porcine melanosomes were coated with the photosensitizer rose Bengal and phagocytized by ARPE-19 cells. Cultures were treated with green light (540-570nm) to generate a sub-lethal photo-chemical reaction located at the site of the phagocytized granules. Phagosomes were re-isolated from cells at intervals after illumination and organelle-associated protein was retrieved and analyzed by Western blotting for proteins known to support motility. Phagosome motility was quantified in paired cultures by live-cell imaging using image capture at 5s intervals over 10min before (baseline) and after illumination.
Phagosome-associated protein extracts contained cytoskeletal proteins actin and tubulin and proteins involved in trafficking along actin microfilaments (e.g., Rab27a) and microtubules (MT) (e.g., dynactin). Also found was the kinase ERK1/2, previously shown to associate with endogenous melanosomes and to regulate their movement. Light treatment produced a dose-dependent loss of phagosome-associated protein that correlated in time with motility slowing; losses were preferentially in proteins mediating MT-based motility. ERK1/2 also dissociated from phagosomes following a transient (1-5 min) change in phosphorylation. Inhibition of ERK by blocking the upstream kinase MEK did not affect baseline phagosome motility but it is not yet known whether light-induced motility slowing is related to ERK phosphorylation.
Impaired motility of phagosomes in the RPE on photic stress may result from dissociation of motor protein complexes and the supporting cytoskeleton from the organelle surface. Since similar motility machinery moves all membrane-bound organelles, a consequence of oxidative stress to the aging RPE may be a general trafficking failure that ultimately disrupts cell phenotype and secondarily impairs RPE support for the retina.
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