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.

Fig. 1.

Immunoblots of membrane proteins of erythroblasts at different stages of differentiation. (A) Transmembrane proteins: blots of SDS/PAGEs of total cellular protein prepared from erythroblasts at 0, 12, 24, and 44 h in culture probed with antibodies against the indicated proteins. Note increased expression of band 3, GPA, Rh, RhAG, CD47, and Duffy and the decreased expression or loss of β1 integrin, CD44, Lu, and ICAM-4 during terminal erythroid differentiation. CD71 and XK increased from proerythroblasts to basophilic erythroblasts with no further changes observed at later stage erythroblasts. (B) Effect of N-glycosidase treatment on Kell and β1 integrin proteins. 0- or 44-h cells in culture either untreated (−) or treated (+) with N-glycosidase were probed with anti-Kell or anti-β1 integrin antibodies. Note the conversion of the 130-kDa Kell band to the 94-kDa Kell band and the reduced size of both β1 integrin bands following N-glycosidase treatment. (C) Skeletal proteins. Blots of SDS/PAGEs of total membrane protein were probed with antibodies against the indicated skeletal proteins. Note the increased expression of all skeletal proteins except for actin during terminal erythroid differentiation. GAPDH was used as loading control for both transmembrane and skeletal proteins.

Fig. 2.

Flow cytometric analysis of expression of transmembrane proteins CD71, GPA, Kell, β1 integrin, and CD44 at different stages of erythroid differentiation. The ordinate measures the number of cells displaying the fluorescent intensity given by the abscissa. Note increased expression of GPA and decreased expression of CD44 during terminal erythroid differentiation.

Fig. 3.

Flow cytometric analysis of bone marrow cells. (A–C) Bone marrow cells labeled with antibodies against TER119 and CD44. (A) plot of CD44 versus TER119. (B) Plot of CD44 versus FSC of all TER positive cells. Note that 5 distinct clusters can be distinguished. (C) The CD44 expression levels in the gated cell population. Note the progressive decrease of CD44 surface expression from region I to region V. (D–F) Bone marrow cells labeled with antibodies against TER119 and CD71. Note that cells from regions I to III express similar levels of CD71.

Fig. 4.

Isolation of erythroblasts at different stages of maturation by cell sorting using CD44 (or CD71), TER119, and FSC as markers. (A) Representative images of erythroblast morphology on stained cytospins from the 5 distinct regions shown in Fig. 3 A and B. (B) Representative images of erythroblast morphology on stained cytospins from the 5 distinct regions shown in Fig. 3 D and E. (C) Quantitation of the purity of erythroblasts at different stages of maturation in various sorted populations using CD44. (D) Quantitation of the purity of erythroblasts at different stages of maturation in various sorted populations using CD71.

Fig. 5.

Comparison of CD44 and CD71 expression in dual-stained bone marrow cells. Primary bone marrow cells were simultaneously stained with Ter-119, CD44, and CD71. (A) Plot of CD44 versus FSC of all TER positive cells. Note that 5 distinct clusters can be distinguished. (B) The CD44 expression levels in the gated cell population. Note the progressive decrease of CD44 surface expression from region I to region V. (C) The CD71 expression levels in the identically gated cell populations. Note the significant overlap of CD71expression. (D) Plot of CD44 versus CD71 of all TER positive cells. Note the progressive decrease of CD44 expression but a broad range of CD71 expression from region I to region V.