How to study heterophagy in RPE cells

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RPE is one of the most active phagocytic cell types in the body, phagocytosing 10% of total photoreceptor volume daily. Phagocytosis of the shed discs is essential to the survival of photoreceptor cells. This phagocytosis process can be divided into four distinct stages: 1) recognition and attachment of the OS; 2) ingestion of the OS; 3) formation of the phagosome and fusion with lysosomes; and 4) finally, digestion. Once OS are internalized by the RPE cells, the phagosome proceeds through a maturation process, followed by fusion with a lysosome to form a phagolysosome where degradation occurs. This pathway remains poorly understood in RPE. We have provided evidence that bA3/A1-crystallin has no apparent role in recognition or ingestion of the OS. However, loss of bA3/A1-crystallin in RPE has an impaired ability to degrade OS, perhaps resulting from a defect in the phagolysosomal pathway. The overall experimental design will be to determine the following parameters by modulating the expression of bA3/A1-crystallin in RPE: (1) Expression levels of proteins involved in phagosome maturation; (2) Interaction of phagocytic vacuoles with early and late endosomes; and (3) Phagosome-lysosome fusion as assessed by live cell imaging

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