Removal of organelles and cytoplasmic material during terminal differentiation of mammalian erythrocytes is an essential and highly regulated process for which autophagy is proposed to play an important role, Autophagosomes have often been described in ultrastructural studies of differentiating erythroid cells in different species, and more recently, genetic deletions of essential autophagy genes in mice support a key role for autophagy in the removal of mitochondria. Despite this, the precise functions of autophagy during erythropoiesis remain obscure. This project explored the roles and regulation of autophagy during human erythropoiesis using an ex vivo culture system. Fluorescence-based imaging of erythroid cells demonstrated that autophagy is activated in this system at the very start of terminal differentiation. Simultaneously, a number of key autophagy genes are transcriptionally upregulated. Using high pressure freezing and electron microscopy, analysis of the kinetics of autophagy induction relative to organelle removal and global cellular remodelling (e.g. nuclear condensation) were carried out. The frequency of autophagosomes increased at the polychromatic stage, meanwhile the endocytic compartment shifted from multivesicular bodies to autophagosomal/endosomal hybrid organelles (amphisomes). Mitochondrial depletion occurred primarily around the time of enucleation. Autophagy can be inhibited by overexpression of catalytically inactive mutants of the endopeptidase, Atg4. To investigate the requirement for autophagy during human erythropoiesis, we used lentiviruses to stably overexpress mutant forms of Atg4B and Atg4D (an Atg4 family member that is dramatically upregulated during human erythropoiesis). The basal number of autophagosomes was significantly reduced in progenitor cells expressing mutant Atg4B (but not in cells expressing mutant Atg4D), but autophagosome frequency was apparently unchanged at later stages of differentiation in either cell-type. Significantly though, both mutants caused an accumulation of large undegraded autophagic compartments, especially at the polychromatic stage, suggesting important roles for Atg4 during autophagosome maturation and/or clearance. Caspase-cleavage of Atg4D can drive mitochondrial import of the cleaved product in erythroid and non-erythroid cells. Further studies showing the potential impact that this has upon mitochondrial ultrastructure, mitochondrial ROS and mitophagy in erythroid cells are also presented.

Human erythrocytes are highly specialised cells boasting numerous features to maximise gas carriage, exchange and delivery around the body. The role of the highly proteinaceous red cell membrane in these processes is vital. Some membrane proteins such as the Rh-associated glycoprotein (RhAG) and aquaporin-1 (AQP1) are postulated to form gas channels, and disorders affecting membrane proteins can have extensive effects on normal red cell function. In this work, the role and interactions of Rh proteins are probed using rare variant Rh-deficient erythrocytes. The RhCE polypeptide is required for normal expression of other members of the Rh complex. Lack of RhCE is associated with depressed expression of CD44, an adhesion molecule, and may alter expression of proteins involved in complement such as decay acceleration factor and Iymphocyte function-associated antigen 3. Absence of RhAG prevents Rh complex expression and is found to affect band 3 macrocomplex proteins GPA and protein 4.2, highlighting the important role for RhAG in the macrocomplex. AQP1 is increased in the absence of RhAG, which supports the hypothesis that they share similar functions. The hereditary stomatocytoses are disorders that affect the ion permeability of red cell membranes. This work comprises a study of the pleiotropic disorder stomatin-deficient cryohydrocytosis (sdCHC), which is caused by mutations in the red cell glucose transporter, glut1. The mutant proteins show minimal glucose transport and increased permeability to cations when expressed heterologously in Xenopus laevis oocytes - consistent with the disease phenotype. sdCHC erythrocytes have very reduced amounts of stomatin, a monotopic membrane protein, a feature shared by other very leaky red cells. This is accompanied by a concomitant increase in stomatin-like protein 2, whose function in red cells is currently unknown. The fates of stomatin proteins in normal and leaky cells are investigated throughout erythropoiesis and found to differ between cation-leaky phenotypes.

EPO/EPOR signalling pathway is essential for the proliferation, differentiation, and survival of erythroid progenitors. Defects in EPO/EPOR signalling regulation cause myeloproliferative disorders, such as erythrocytosis, or polycythemia vera. Erythrocytosis is a disorder associated with truncations of the human EPOR resulting in deletions of 59-110 amino acids from the cytoplasmic tail of EPOR. The transformation capacity of JAK2V617F, a mutation responsible for myeloproliferative neoplasias is dependent on the presence of EPOR. Therefore, the elucidation of the mechanisms controlling EPO/EPOR signalling and proliferation/differentiation of erytlu'oid progenitors attracts interest. Ubiquitination and degradation is a common regulatory mechanisms affecting signalling from a variety of receptors. SOCS proteins regulate receptor signalling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. The aim of this study is to clarify the role of lysines important for EPOR ubiquitination in regulating EPO/EPOR f . signalling, erythroid cell proliferation and role of SOCS3 as ubiquitin ligase. Here, it is shown that K428 plays a key role in EPOR routing and demonstrate that the effects of SOCS3 on EPO/EPOR signalling depend on K428. Mutating K428 to arginine (K428R) blocked EPOR ubiquitination and enhanced stabilisation of the mature form of EPOR K428R in comparison to the wild-type receptor. This also leads to accumulation of EPOR in late endosomes and leads to sustained activation of ST AT5 and hypersensitivity to EPO leading to increased proliferation. SOCS3 was observed to ubiquitinate EPOR on this lysine and is important for trafficking the receptor from late endosomes to lysosomes. These findings clearly show that K428 is crucial for EPOR ubiquitination and attenuation of EPO responses. It was also observed JAK2V617F escapes SOCS3 inhibition by accumulating EPOR in late endosomes and utilising it for the constitutive signalling for transformation of the cells. This is the first demonstration of SOCS-mediated ubiquitination by targeting K428 and routing of EPOR to lysosomes.

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L’érythropoïèse est le processus aboutissant à la production des hématies à partir d’une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d’expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l’ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu’en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l’imatinib, les érythroblastes cultivés ex vivo perdent l’expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l’érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l’imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l’imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l’imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l’imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l’imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l’imatinib lève l’auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L’étude de la taille et du potentiel prolifératif des cellules, ainsi que l’analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d’un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), « matrice » du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l’évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu’au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d’internalisation d’un récepteur à activité tyrosine kinase de classe III, c-Kit par l’imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque. / Erythropoiesis is the process leading to the production of red blood cells from hematopoietic stem cell. The erythroid differentiation involves morphological cell changes, in part related to the loss of membrane expression of the type III receptor tyrosine kinase, c-Kit. In response to its ligand SCF, c-Kit is activated, then internalized and degraded by the proteasome pathway via the E3 ubiquitin ligase c-Cbl, or by the lysosomal pathway, after endocytosis. In the first part of this work, we demonstrated that in the absence of SCF and in response to tyrosine kinase inhibitor, imatinib, erythroblasts cultured ex vivo, lose membrane expression of c-Kit and accelerate their terminal differentiation. In view of these observations, we sought to understand the mechanisms involved. On an erythropoietin dependent cell line expressing c-Kit at the membrane, we showed that imatinib induces receptor internalization and degradation by the lysosomal pathway, independently of c -Cbl. Furthermore, we showed that this effect is reversible and that imatinib does not block the c-Kit re-expression after its internalization, in response to SCF. Metabolic labelling showed that imatinib does not alter synthesis or maturation of c -Kit and that the phospho-tyrosine profile of cells treated with imatinib is generally unchanged. Finally, we showed that the binding of imatinib to the catalytic pocket of c-Kit is essential for its internalization, and therefore its degradation. So, it appears that imatinib removes c-Kit self-inhibition, which seems necessary to its retention at the membrane. In the second part of this work, we studied the morphological changes of nucleoli, the site of ribosome biogenesis, during erythroid differentiation. We showed that the reduction of cell size takes place at the same time than reduction of cell proliferation and reduction of surface and volume of the Granular Compound (GC), the “matrix” of the nucleolus. Moreover, we showed by electronic microscopy, the persistence of GC at the end of maturation. Finally, we also studied the evolution of nucleoli in a pathological context of low risk myelodysplastic syndromes, which are characterized by ineffective hematopoiesis. We observed that immature pathological cells have larger GC than immature normal cells, but that during differentiation, the morphology of nucleoli is identical between normal and pathological cells. In conclusion, this work has allowed us to describe 1) the mechanisms of internalization of a class III receptor tyrosine kinase, c-Kit by imatinib and 2) the morphology of the nucleolus during normal and pathological low risk myelodysplastic syndromes of erythroid differentiation.