A particularly intriguing aspect of the erythroblast enucleation process, which creates an anucleate immature erythrocyte called a reticulocyte, is how the sorting of proteins between the nascent reticulocyte and the extruding nucleus occurs. A large-scale proteomic analysis was conducted on isolated pure populations of reticulocytes and extruded nuclei, and the results for key proteins of interest confirmed using western blotting. Many erythroid membrane and cytoskeletal proteins partitioned predominantly or exclusively to the reticulocyte. In contrast, nuclear proteins, endoplasmic reticulum proteins and a contingent of membrane proteins were enriched in the extruded nuclei. This confirms enucleation as a key remodelling event in the development of an erythrocyte where important proteins needed for the erythrocyte are retained, but unwanted or excess proteins are removed with the nucleus. During this thesis lentiviral transduction methodology was further developed to achieve both knockdown of cytoskeletal proteins and overexpression of cytoskeleton fragments to disrupt the erythroid cytoskeleton and elucidate the role membrane-cytoskeletal interactions play in membrane multi-protein complex assembly and stabilisation during erythropoiesis. shRNA mediated knockdown of ankyrin achieved the most striking results with >95% reduction in ankyrin levels. The expression profiles of key erythroid membrane and cytoskeletal proteins following ankyrin knockdown were monitored throughout terminal differentiation by flow cytometry and western blotting. Ankyrin is required for the stabilisation of members of the ankyrin complex throughout terminal differentiation, and these dependencies become established at different stages for different members within the complex. The rhesus subcomplex members Rh, RhAG and CD47 were depleted from the basophilic erythroblast stage in the absence of ankyrin, with additional loss during enucleation. Protein 4.2 also exhibited early depletion at the basophilic stage. Interestingly, band 3 and GPA levels were comparable to the control throughout the early stages of terminal differentiation when ankyrin was depleted, but then exhibited decreased retention with the reticulocyte during enucleation. RhAG loss at the basophilic stage was shown to be due to enhanced internalisation and turnover. The work presented here has greatly increased our understanding of the assembly of membrane multi-protein complexes and the protein sorting processes that occur during human terminal differentiation and enucleation, and has provided an explanation for the secondary protein deficiencies observed in Hereditary Spherocytosis where the attachment of membrane proteins in the bilayer to the underlying cytoskeleton is disrupted.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:685916 |
| Date | January 2015 |
| Creators | Bell, Amanda Jayne |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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