In mammals, erythropoiesis takes place in distinct anatomic units called erythroblastic islands that are consisted of a central macrophage surrounded by erythroblasts of varying maturity. The attachment of erythroblasts to macrophages within these islands promotes terminal maturation and enucleation of erythroid cells. Among a few other adhesion molecules known to date, Emp (erythroblast macrophage protein) is involved in this association. Our recent gene targeting studies showed that in the absence of Emp, erythroblastic islands are not formed and erythroid cells do not undergo enucleation. In addition, Emp null macrophages do not fully mature. Information on the changes in the expression level and cellular localization of Emp in differentiating erythroid and macrophage cells is essential for understanding the function of Emp both in the formation of erythroblastic islands and in the development of these two cell types. Previously we showed that Emp is expressed in immature erythroid precursors but not in mature erythrocytes, consistent with the fact that mature red blood cells do not adhere. However, no information is available on the expression of Emp in macrophages. To address this issue, we have used primary mouse fetal liver macrophages cultured for various time periods. Our studies showed that while Emp was expressed in all stages of maturation, the localization pattern changed dramatically during maturation: in immature macrophages, a substantial fraction of Emp was intracellular, whereas in more mature cells, Emp was expressed largely at the plasma membrane. To determine if the membrane-associated Emp is exposed on the cell surface, we employed biotin labeling of surface proteins, and compared the fraction of total Emp that is accessible to the biotinylation reagent with the fraction that is inaccessible. Our data showed that approximately 5–20% of total cellular Emp was present on the surface of immature macrophage precursors compared to 60–70% in fully matured macrophages. Cell surface expression of Emp was further confirmed by live cell staining, without prior fixation, of macrophages at different stages of maturation. Intracellular pool of Emp was present largely in the nucleus where it co-localized with the nuclear matrix marker, the spliceosome assembly factor SC35. To examine the trafficking of newly synthesized Emp, we performed pulse-chase experiments in macrophages of varying maturity. We found that nascent Emp migrated intracellularly from the nucleus to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture showed that erythroid cells attached to mature macrophages but not to immature macrophage precursors. Taken together, our data shows that the temporal and spatial expression of Emp in erythroid and myeloid cells correlates with its involvement in the attachment of these two cell types in mammalian erythropoiesis. Furthermore, localization of Emp at multiple sites within the cellular environment suggests functions not limited to cell attachment.

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