The reticulocyte (retic) is the immediate precursor of a red blood cell (RBC). It lacks the nucleus (in mammals) and is still immature, because it has to reduce its size by losing surface area and volume, before attaining the characteristic shape of an RBC; the biconcave discocyte. This whole process is achieved in two steps. The first takes place in the bone marrow, where the R1 retics activate endogenous mechanisms for the: i) polyubiquitylation/proteasome-mediated degradation of no longer needed intracellular proteins; ii) autophagy of intracellular organelles and iii) a vesicle-based mechanism for the reduction of membrane surface area and the selective removal of certain membrane proteins. The second step begins when the R1 retics are released from the marrow into the general circulation as R2 retics. Little is known of the mechanisms involved in the maturation of circulating R2 retics. It is in particular unknown whether this is a spontaneous and selective process such as that responsible for the maturation of R1 retics. The problem is relevant in the field of medical biotechnology: it is nowadays possible to cultivate in vitro erythroid precursors and to differentiate them to the stage of anucleated reticulocytes on a large scale with potential application in transfusion medicine. However, full maturation in vitro of retics to discocytic RBCs is still a major challenge. In the course of our work, we tried to understand the problem behind the terminal maturation of R2 retics at the molecular level. In fact, during R1 maturation, exosomes are released and they were found to contain the transferrin receptor (TfR) and membrane rafts but no band 3 and spectrin (respectively, the most abundant transmembrane and membrane-skeletal proteins of the RBC). Therefore, we wanted to verify if this same observation was true for the R2 maturation process, by analyzing the levels of band 3 and spectrin in samples of pure R2 retics and mature RBCs from the same donors. The purification of populations of circulating retics having a different degree of maturation was done by positive immune-magnetic sorting. The results obtained demonstrated that the contents of band 3 and spectrin, which are conserved all through R1 retic maturation, decrease, on a per-cell basis, in the transformation process from R2 to RBCs. In previous studies we demonstrated that membrane rafts are retained during maturation of R2 retics. These results are quite intriguing, as in the R1 maturation step band 3 and spectrin are completely retained in the cell, whereas membrane rafts are selectively lost with the exosomes. We have a quite clear indication that different, and apparently diametrically opposed, mechanisms are at the basis of the maturation of the two types of retics. The final maturation of the R2 retic in the circulation cannot be driven by intrinsic programs, as the cell is now devoid of the necessary molecular machinery, but it requires a systemic intervention by other organs. It is most likely the spleen, liver, and/or the endothelium that actively process this cell type that is no longer endowed with spontaneous mechanisms for attaining the final size and shape of a mature RBC. Exploring the basic mechanisms underlying the membrane remodeling of retics that lead to their complete maturation to RBCs, could help find the right culture conditions in vitro and potentially revolutionize transfusion medicine.
IL PROBLEMA DEL DIFFERENZIAMENTO TERMINALE DEI RETICOLOCITI UMANI AI GLOBULI ROSSI CIRCOLANTI MATURI
THE PROBLEM OF THE TERMINAL DIFFERENTIATION OF HUMAN RETICULOCYTES TO MATURE CIRCULATING RED BLOOD CELLS
PELEU NGATE, ALAIN PATRICK
2020/2021
Abstract
The reticulocyte (retic) is the immediate precursor of a red blood cell (RBC). It lacks the nucleus (in mammals) and is still immature, because it has to reduce its size by losing surface area and volume, before attaining the characteristic shape of an RBC; the biconcave discocyte. This whole process is achieved in two steps. The first takes place in the bone marrow, where the R1 retics activate endogenous mechanisms for the: i) polyubiquitylation/proteasome-mediated degradation of no longer needed intracellular proteins; ii) autophagy of intracellular organelles and iii) a vesicle-based mechanism for the reduction of membrane surface area and the selective removal of certain membrane proteins. The second step begins when the R1 retics are released from the marrow into the general circulation as R2 retics. Little is known of the mechanisms involved in the maturation of circulating R2 retics. It is in particular unknown whether this is a spontaneous and selective process such as that responsible for the maturation of R1 retics. The problem is relevant in the field of medical biotechnology: it is nowadays possible to cultivate in vitro erythroid precursors and to differentiate them to the stage of anucleated reticulocytes on a large scale with potential application in transfusion medicine. However, full maturation in vitro of retics to discocytic RBCs is still a major challenge. In the course of our work, we tried to understand the problem behind the terminal maturation of R2 retics at the molecular level. In fact, during R1 maturation, exosomes are released and they were found to contain the transferrin receptor (TfR) and membrane rafts but no band 3 and spectrin (respectively, the most abundant transmembrane and membrane-skeletal proteins of the RBC). Therefore, we wanted to verify if this same observation was true for the R2 maturation process, by analyzing the levels of band 3 and spectrin in samples of pure R2 retics and mature RBCs from the same donors. The purification of populations of circulating retics having a different degree of maturation was done by positive immune-magnetic sorting. The results obtained demonstrated that the contents of band 3 and spectrin, which are conserved all through R1 retic maturation, decrease, on a per-cell basis, in the transformation process from R2 to RBCs. In previous studies we demonstrated that membrane rafts are retained during maturation of R2 retics. These results are quite intriguing, as in the R1 maturation step band 3 and spectrin are completely retained in the cell, whereas membrane rafts are selectively lost with the exosomes. We have a quite clear indication that different, and apparently diametrically opposed, mechanisms are at the basis of the maturation of the two types of retics. The final maturation of the R2 retic in the circulation cannot be driven by intrinsic programs, as the cell is now devoid of the necessary molecular machinery, but it requires a systemic intervention by other organs. It is most likely the spleen, liver, and/or the endothelium that actively process this cell type that is no longer endowed with spontaneous mechanisms for attaining the final size and shape of a mature RBC. Exploring the basic mechanisms underlying the membrane remodeling of retics that lead to their complete maturation to RBCs, could help find the right culture conditions in vitro and potentially revolutionize transfusion medicine.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13137