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Within the context of erythropoiesis and the possibility of producing artificial red blood cells (RBCs) in vitro, a most critical step is the final differentiation of enucleated erythroblasts, or reticulocytes, to a fully mature biconcave discocyte, the RBC. Reviewed here is the current knowledge about this fundamental maturational process. By combining literature data with our own experimental evidence we propose that the early phase in the maturation of reticulocytes to RBCs is driven bya membrane raft-based mechanism for the sorting of disposable membrane proteins, mostly the no longer needed transferrin receptor (TfR), to the multivesicular endosome (MVE) as cargo of intraluminal vesicles that are subsequently exocytosed as exosomes, consistently with the seminal and original observation of Johnstone and collaborators of more than 30 years ago (Pan BT, Johnstone RM. Cell. 1983;33:967-978). According to a strikingly selective sorting process, the TfR becomes cargo destined to exocytosis while other molecules, including the most abundant RBC transmembrane protein, band 3, are completely retained in the cell membrane. It is also proposed that while this process could be operating in the early maturational steps in the bone marrow, additional mechanism(s) must be at play for the final removal of the excess reticulocyte membrane that is observed to occur in the circulation. This processing will most likely require the intervention of the spleen, whose function is also necessary for the continuous remodeling of the RBC membrane all along this cell's circulatory life. [ABSTRACT FROM AUTHOR]

Background/Aims: A high surface-to-volume ratio and a spectrin membrane-skeleton (MS) confer to the mammalian red blood cells (RBCs) their characteristic deformability, mechanical strength and structural stability. During their 120 days of circulatory life in humans, RBCs decrease in size, while remaining biconcave disks, owing to a coordinated decrease in membrane surface area and cell water. It is generally believed that part of the membrane is lost with the shedding of spectrin-free vesicles of the same type that can be obtained in vitro by different treatments. If this were true, an excess of MS would arise in old RBCs, with respect to the lipid bilayer. Aim of this paper was to investigate this aspect. Methods: Quantification of spectrin by electrophoretic methods was carried out in RBCs of different age. Results: Spectrin decreases, on a per cell basis, with RBC ageing. On the other hand, the membrane raft protein marker flotillin-2, while decreasing in the membrane of old cells, was found to be strongly depleted in the membrane of in vitro-induced vesicles. Conclusion: Part of the membrane-skeleton is probably lost together with part of the lipid bilayer in a balanced way. These findings point to a mechanism for the in vivo release of membrane that is different from that which is known to occur in vitro. [ABSTRACT FROM AUTHOR]

The cell type of election for the study of cell membranes, the mammalian non-nucleated erythrocyte, has been scarcely considered in the research of membrane rafts of the plasma membrane. However, detergent-resistant-membranes (DRM) were actually first described in human erythrocytes, as a fraction resisting solubilization by the nonionic detergent Triton X-100. These DRMs were insoluble entities of high density, easily pelleted by centrifugation, as opposed to the now accepted concept of lipid raft-like membrane fractions as material floating in low-density regions of sucrose gradients. The present article reviews the available literature on membrane rafts/DRMs in human erythrocytes from an historical point of view, describing the experiments that provided the solution to the above described discrepancy and suggesting possible avenue of research in the field of membrane rafts that, moving from the most studied model of living cell membrane, the erythrocyte's, could be relevant also for other cell types. [ABSTRACT FROM AUTHOR]

Abstract: Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy. [Copyright &y& Elsevier]

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4 degrees C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C12E8. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C12E8, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C12E8 in comparison to intact cells, while the difference in the S values between Triton X-100 and C12E8 DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C12E8 detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs. [ABSTRACT FROM AUTHOR]

Abstract: Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane skeleton in human erythrocytes and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils that lead to proteolytic disruption of the supramolecular assembly of the membrane skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions that most likely involve the spectrin molecule. [ABSTRACT FROM AUTHOR]