Metcalf, D.J.;
(2009)
The cellular machinery that underpins recycling
through the Trans-Golgi Network.
Doctoral thesis , UCL (University College London).
Abstract
Retromer is a pentameric complex required for endosome to TGN transport of transmembrane cargo, such that in mammalian cells a lack of retromer function leads to a mannose-6-phosphate receptor defect in the delivery of hydrolases to lysosomes. CLN3 is a multipass membrane protein that is localised throughout the post- Golgi trafficking system. Mutations cause an autonomic recessive early-onset neurodegenerative disorder called juvenile Batten disease. The most severe form of Batten disease is caused by a mutation in cathepsin D, a lysosomal hydrolase. When the yeast orthologue of CLN3, BTN1, is deleted BTN2 is upregulated. BTN2 has been shown to mediate endosome to TGN trafficking of some cargo raising the possibility that this pathway is defective in patients. An siRNA approach was undertaken to investigate the role of CLN3 in mannose phosphate receptor trafficking. P-selectin is a leukocyte receptor expressed in endothelial cells which functions early in the inflammatory cascade. It is stored within the endothelial storage organelle, Weibel-Palade bodies (WPBs). P-selectin traffics to the cell surface via exocytosing WPBs and is rapidly internalised into endosomes. Subsequently, it can take a degradative route to lysosomes or a recycling route to the trans-Golgi network (TGN). The cytoplasmic tail of P-selectin interacts with sorting nexin 1 which is a component of the retromer complex. An siRNA approach was undertaken to investigate the role of retromer function in P-selectin recycling to the TGN. In HUVEC a partial knockdown was achieved, however, no change in P-selectin trafficking was detected. In non-endothelial cell systems the P-selectin antibody uptake and immunofluorescence assay was unable to resolve TGN recycling. A defect was found in the trafficking of an MPR reporter protein after CLN3 depletion using siRNA. The MPR reporter protein was further analysed and found to be redistributed to the TGN. Further trafficking analyses ruled out defects in clathrin- mediated endocytosis or endocytic to lysosomal or endocytic to TGN trafficking. By developing a FRAP approach using a GFP-MPR tracer protein a reduction in TGN exit was found along with a reduction in cathepsin D lysosomal delivery. Further investigations indicate that CLN3 is not mediating this effect by disrupting AP-1 function.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | The cellular machinery that underpins recycling through the Trans-Golgi Network |
| Language: | English |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/14728 |
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