Terminal maturation of human reticulocytes to red blood cells by extensive remodelling and progressive liquid ordering of membrane lipids

Abstract

ABSTRACTThe terminal differentiation of reticulocytes to mature red blood cells (RBCs) in mammals is a topic of intense investigation. Although much has been learned about the early phase of reticulocyte maturation in the bone marrow of the adult mammal, after enucleation of the orthochromatic erythroblast, the final maturation of peripheral circulating reticulocytes into RBCs is still poorly understood. Research on reticulocyte maturation was predominantly carried out on stress reticulocytes, or mixed populations of RBCs and normal reticulocytes, strongly limiting the possibility of discriminating different maturational phases and the relative underlying mechanisms. Another limitation was a somewhat “proteinocentric” approach, whereby possible qualitative and quantitative changes in the most important structural component of the membrane, the lipids, were not investigated.In the marrow, the reticulocyte matures through processes intrinsic to the cell (autophagy, ubiquitin/proteasome-dependent proteolysis, exosome release, regulated volume decrease) to reduce its size and simplify its composition. In the circulation, the task is taken over by systemic intervention that is necessary for the removal of extra surface area and to stimulate further volume decrease. The last two processes are preliminary to and go along with the completion of the assembly of the membrane skeleton and its anchoring to the bilayer into a structurally and functionally stable plasma membrane. The selective membrane removal in the marrow phase of maturation leads to the loss of most of the transferrin receptor through release of exosomes enriched in membrane rafts. In the circulatory phase, membrane area decrease is obtained by (a) different mechanism(s), as indicated by several pieces of evidence, including the results of the present studyWe have isolated here two populations of circulating reticulocytes at different levels of maturation in vivo, and three subpopulations of RBCs of different age from normal human donors, and characterized the evolution of their lipidome. Sphingomyelin, cholesterol and in part phosphatidylethanolamine increase in relative terms, whereas phosphatidylcholine and phosphatidylserine decrease from immature reticulocytes to mature RBCs, at the same time as the surface area per cell decreases. Moreover, the relative amounts of the more than 70 phospholipid subclasses evaluated in the study, based on the number of carbon atoms (12-24) and of double bonds (0-6) in the fatty acids linked to the phospholipid, also change in the process. This complex remodeling suggests the presence of an underlying blueprint, whereby changes in each individual phospholipid subclass all seem to converge in producing a state of higher liquid order, and thus of higher stability, in the lipid bilayer. Results also strongly hint at the lipid remodeling as the driving force, together with cell shrinkage, for the organization of spectrin in an extended meshwork and its anchoring to the bilayer through vertical interactions with intrinsic membrane proteins. A model is proposed, based on concepts of lipid interdigitation and protein “pinning”.