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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:566696 |
| Date | January 2012 |
| Creators | Betin, Virginie M. S. |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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