The retinal pigment epithelium (RPE) is a monolayer of highly polarised cells that lies between the photoreceptors and choriocapillaris and performs a crucial role in the maintenance of visual function. The RPE phagocytoses and digests shed photoreceptor outer segments, transports nutrients, ions and water and secretes various essential growth factors that support surrounding cells. Despite the importance of membrane traffic pathways in the RPE little is known about endocytosis in these cells. Furthermore, although the early stages of phagocytosis of photoreceptor outer segments have been well characterised, the processing of the phagosome after engulfment is poorly understood. The first aim of this PhD project was to characterise the organisation of the endocytic pathway in the RPE and identify potential endocytic cargos that could be used to monitor endocytosis in RPE cells in culture. The second aim was to identify methods of characterising sequential stages of phagosome maturation in the RPE in situ and determine whether the same stages could be reproduced in cultured RPE cells. The final aim was to identify mechanisms underlying phagosome maturation, focusing in particular on the role of interactions with the endocytic pathway. Endocytic compartment markers and potential endocytic cargos were localised in situ on retinal sections and in vitro using primary porcine RPE cells. In situ and in vitro studies showed that Rab11a, a marker for apical recycling endosomes, is found distributed throughout the cell and not restricted to an apical compartment as seen in other epithelial cells. Transferrin receptors are expressed on both apical and basal plasma membranes in the RPE and both fluid phase probes and transferrin endocytosed from apical and basal surfaces meet in a common endocytic compartment. Phagosome maturation was investigated by immuno-electron microscopy using antibodies to two different rhodopsin epitopes. Loss of a C-terminal cytoplasmically exposed epitope was an early step in phagosome maturation, occurring before phagosome:lysosome fusion, which allowed the distinction of early and late phagosomes. Here it is demonstrated that the same stages of phagosome maturation occurred, albeit more slowly, in cultured porcine RPE challenged with isolated porcine outer segments. Treatment with the protease inhibitor, leupeptin, inhibited loss of the C-terminal rhodopsin epitope, suggesting that limited proteolysis can occur within the maturing phagosome. Finally, by loading the endocytic pathway from the basal surface and the phagocytic pathway from the apical surface it was possible to demonstrate acquisition of endocytic content by the maturing phagosome, the timing of which suggests that interaction with the endocytic pathway is likely to be a factor in the limited proteolysis of rhodopsin that occurs in the maturing phagosome. This work opens the possibility to investigate defects in endocytic and phagocytic pathways in retinal disease and determine how these defects could lead to phenotypes associated with aging and retinal degeneration.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626158 |
| Date | January 2013 |
| Creators | Porpino Meschede, I. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1391573/ |
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