Membrane Rearrangements in the Maturation of Circulating Human Reticulocytes

Abstract

Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a "per cell" basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC.

Interplay Between Plasma Membrane Lipid Alteration, Oxidative Stress and Calcium-Based Mechanism for Extracellular Vesicle Biogenesis From Erythrocytes During Blood Storage

“…For instance, R1 reticulocytes undergo significant rearrangements in their membrane and intracellular components via several mechanisms including exosome release. Circulating R2 reticulocytes complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins ( Minetti et al, 2018 , 2020 ). Membrane remodelling and vesicle formation also occur during RBC ageing ( Ciana et al, 2017b ).…”

Impaired Cytoskeletal and Membrane Biophysical Properties of Acanthocytes in Hypobetalipoproteinemia – A Case Study

“…Actually, the resistance to PS surface exposure has also been reported by Siegl et al (2013) in chorea-acanthocytosis after RBC stimulation with lysophosphatidic acid. Thus, pHypoβ has a high proportion of acanthocytes which could result from a maturation defect during the R1 reticulocyte stage known to undergo significant rearrangements in reticulocyte membrane and intracellular components via several mechanisms including exosome release and mitophagy (Minetti et al, 2018(Minetti et al, , 2020.…”

Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis

“…Accordingly, SDS-PAGE/western blotting evidenced that the α- and β-spectrin-to-Band3 ratios were not modified in pEl vs. healthy donors whereas the Band7-to-Band3 was decreased by ~30% (data not shown). Those data indicated that protein modifications in pEl did not result from the higher reticulocyte content since reticulocyte membranes instead show higher β-spectrin- and Band7-to-Band3 ratio as compared to mature RBCs [ 32 ]. The cytoskeleton:membrane occupancy was also preserved in pEl RBCs, in contrast to stored RBCs used as positive control, but its distribution was more heterogeneous ( Figure 2 A–C and Figure S3A ), showing spectrin gathering at one cell edge in ~75% of pEl RBCs vs. ~20% in pElm RBCs and ~5% in healthy RBCs (data not shown).…”

“…Phosphatidylcholine and PS species, in particular those having long and unsaturated fatty acids, were strongly reduced as compared to a series of healthy donors ( Figure 4 E,G and Figure S8A–D ). Such lipid modifications were not due to the higher reticulocyte content in pEl since cholesterol and sphingomyelin have been shown to instead decrease and phosphatidylcholine and PS species to increase in reticulocytes vs. mature RBCs [ 32 ]. They could be related to the decreased content in very long chain fatty acid elongation protein 2 ( Figure S6B ), but also, in the case of PS, to the strong increase in several monoacyl lysoPS derivatives ( Figure 4 F,H).…”

“…The increased membrane ghost association of tubulin β6 and components of the protein biosynthesis/folding machinery vs. decreased Rh(D) polypeptide suggested aberrant membrane defects already during erythropoiesis [ 41 , 48 , 49 ]. Although this remains to be proved, this hypothesis is supported by the fact that membrane rearrangements during erythropoiesis are necessary to generate normal mature RBCs [ 32 ].…”