AbstractThe process of reticulocyte maturation, whereby the reticulocyte undergoes differentiation to become a fully mature erythrocyte, occurs after egress of the cell from the bone marrow and into circulation. Reticulocyte maturation is known to require extensive intracellular remodelling, which is further characterised by loss of cell volume, removal of redundant proteins and cell organelles and highly selective membrane and cytoskeletal rearrangement.
However, the specific mechanisms that drive this process in vivo are poorly understood. Furthermore, at present, reticulocytes derived through in vitro culture fail to generate mature erythrocytes in ex vivo conditions. Thus, this project aims to contribute to the understanding of reticulocyte maturation by investigating how the membrane and specialised cytoskeleton remodel to form the final recognisable biconcave red blood cell.
To achieve this, we first use high-throughput proteomic methods to compare in vitro-derived reticulocytes, endogenous reticulocytes and erythrocytes. Concurrently, an ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus induces a decrease in cell volume, which is characterized by differential reduction in protein abundance and increased mitochondrial clearance. Through the two previous approaches we identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA), as exhibiting differential phosphorylation status and global abundance between reticulocytes and erythrocytes and, using a specific inhibitor for its activity alongside the ex vivo circulation system, demonstrate its importance in vesicle loss during reticulocyte remodelling.
Through the combination of a microsphere filtration assay, microfluidics and phosphoproteomics, we then derive a comprehensive and integrative phosphoproteomic dataset for further mechanistic dissection of the cell’s response to deformation. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases RBC capacity to undergo repeated deformation.
Finally, we build a machine learning-based system for the diagnosis of rare anaemias, achieving a maximum prediction accuracy of 92%. During this process, we identify hereditary xerocytosis patients as displaying delayed reticulocyte maturation and show that such a phenotype can be recapitulated through pharmacological treatment with Yoda1, a specific activator of PIEZO1.
In summary, the work developed in this thesis provides a comprehensive, multidisciplinary and collaborative approach to the study of reticulocyte maturation and lays the foundations for substantial future studies in the field.
|Date of Award||23 Jan 2020|
|Supervisor||Ash M Toye (Supervisor) & Jan Frayne (Supervisor)|
- Red blood cells