Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis

Abstract

Erythropoiesis is the process by which nucleated erythroid progenitors proliferate and differentiate to generate, every second, millions of nonnucleated red cells with their unique discoid shape and membrane material properties. Here we examined the time course of appearance of individual membrane protein components during murine erythropoiesis to throw new light on our understanding of the evolution of the unique features of the red cell membrane. We found that the accumulation of all of the major transmembrane and all skeletal proteins of the mature red blood cell, except actin, accrued progressively during terminal erythroid differentiation. At the same time, and in marked contrast, accumulation of various adhesion molecules decreased. In particular, the adhesion molecule, CD44 exhibited a progressive and dramatic decrease from proerythroblast to reticulocyte; this enabled us to devise a new strategy for distinguishing unambiguously between erythroblasts at successive developmental stages. These findings provide unique insights into the genesis of red cell membrane function during erythroblast differentiation and also offer a means of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes.E rythropoiesis is the process by which erythroid progenitors proliferate and differentiate into nonnucleated reticulocytes. Two distinct erythroid progenitors have been functionally defined in colony assays, namely, the early-stage burst-forming uniterythroid (BFU-E) and the later stage colony-forming uniterythroid (CFU-E) progenitor (1). The earliest morphologically recognizable erythroblast in hematopoietic tissues is the proerythroblast, which undergoes 3-4 mitoses to produce reticulocytes. Morphologically distinct populations of erythroblasts are produced by each successive mitosis, beginning with proerythroblasts and followed by basophilic, polychromatic, and orthochromatic erythroblasts. Finally, orthochromatic erythroblasts expel their nuclei to generate reticulocytes. This ordered differentiation process is accompanied by decreases in cell size, enhanced chromatin condensation, progressive hemoglobinization, and marked changes in membrane organization.During recent decades, detailed characterization of the protein composition and structural organization of the mature red cell membrane has led to insights into its function (2-6). A 2-dimensional spectrin-based skeletal network consisting of ␣-and ␤-spectrin, short actin filaments, protein 4.1R, ankyrin, protein 4.2, p55, adducin, dematin, tropomyosin, and tropomodulin has been shown to regulate membrane elasticity and stability. Mutations in some of these proteins result in loss of mechanical integrity and hemolytic anemia. The skeletal network is attached to the lipid bilayer through 2 major linkages (7). The first is through ankyrin, which itself forms part of a complex of band 3, glycophorin A, RhAG, CD47, and ICAM-4, while the second involves protein 4.1R, glycophorin C, and protei...

Lysine-specific demethylase 1 restricts hematopoietic progenitor proliferation and is essential for terminal differentiation

“…Gfi1b expression is normally downregulated during terminal erythropoiesis. 22 Thus, downregulation of GFP and the erythroblast immaturity marker, CD44, 34 were expected to proceed in parallel. Remarkably, GFP downregulation proceeded in parallel with decreasing CD44 expression in LSD1-kd erythroblasts, but from a higher level of GFP expression (Figure 7c).…”

Hematopoietic deletion of transferrin receptor 2 in mice leads to a block in erythroid differentiation during iron‐deficient anemia

“…The cells were then stained with Ter119-fluoroscein-isothiocyanate (FITC) (1:100) (Milteyni Biotec, Bergisch Gladbach, Germany), CD44-allophycocyanin (APC) (1:100) (Biolegend, San Diego, CA) and EPOR-phycoerytherin (PE) (1:100) (Sino Biological Inc, Beijing, China) for 1 hr. The stained single cell suspensions were either analyzed using the LSR Fortessa 4 cytometer (BD Biosciences, Franklin Lakes, NJ) or sorted into five different populations as described previously [29]. The data plots and graphs were generated using the FlowJo software package (Tree Star Inc., Ashland, OR).…”

“…The cells were stained with TER119, an erythroid marker, and CD44, another cell surface marker used for separating differentiating erythroblasts into their five stages of development [29]. An analysis of erythroid cells (TER119-positive cells) revealed that both the BM and spleen of the KO mice fed an ID diet had significantly increased numbers of immature erythroid cells (Bottom right panels in Fig.…”

CD44 in hematological neoplasias

“…Likewise in adult erythropoiesis, decreasing CD44 expression is found. Therefore the authors claim CD44 to be a better marker for discrimination between erythroblasts at different stages of development than CD71 [74].…”