CD47 Is Dependent on the Actin Cytoskeleton for Its Membrane Stability Prior to Protein 4.2 Expression during Early Erythroblast Differentiation

CD47 is a ubiquitously expressed ‘Marker of Self’ that protects cells from phagocytosis, through recognition by SIRPα on macrophages (Oldenborg et al Science 2000). CD47 was originally isolated on ovarian tumour cells (Poels et al J Natl Cancer Inst 1986) and has subsequently been detected on leukemic stem cells, where increased CD47 levels ensure immune evasion (Jaiswal et al Cell 2009). CD47 is also a ‘Marker of Self’ on red cells, but is reduced at the cell surface in certain patients with Hereditary Spherocytosis. In red cells, ~60% of CD47 is connected to the cytoskeleton (Dahl et al Blood 2004). Cytoskeletal connectivity of CD47 in the red cell membrane is dependent on the band 3 complex associated protein 4.2, demonstrated by an ~80% reduction in CD47 levels in protein 4.2 null red cells (Mouro-Chanteloup et al Blood 2003). Previous work (van den Akker et al Haematologica 2009) established that CD47 becomes dependent on protein 4.2 at the basophilic erythroblast stage (48 hours post-differentiation), but it is unknown what interactions support CD47 membrane stability prior to protein 4.2 expression during expansion and early erythroid differentiation. CD47 mRNA is alternatively spliced giving rise to four potential isoforms. The most abundant isoforms are form 2, expressed in all bone-marrow derived cells, and form 4 (and form 3), found predominantly in neural tissues (Reinhold et al J Cell Sci 1995). CD47 isoform 2 is the only form expressed on mature red cells, but we hypothesized that expression of other CD47 isoforms with different trafficking or binding characteristics could explain the independence of CD47 prior to band 3 complex assembly. Using specific polyclonal antibodies to multiple CD47 isoforms, we demonstrate that isoform 2 is expressed prior to and throughout in vitroerythroid differentiation. CD47 isoforms 3 and 4 were detected by western blotting until the late polychromatic erythroblast stage (96 hours post-differentiation), but only CD47 isoform 2 was detected at the cell surface. Therefore, we next hypothesised that CD47 must interact with another protein or exhibit different trafficking characteristics to maintain its membrane stability early during terminal differentiation. To identify a candidate protein or associated protein complex, CD47 was immunoprecipitated from expanding erythroblasts (Exp), proerythroblasts (T0), and basophilic erythroblasts (T48), and analysed via Nano-LC mass spectroscopy. In Exp and T0 erythroblasts, CD47 pulled down actin and multiple actin-associated proteins. These interactions were not observed in T48 erythroblasts, corresponding to the time during terminal differentiation when CD47 is dependent on protein 4.2. To confirm a dependence on actin for CD47 membrane stability, well-characterised drugs that disrupt actin dynamics were employed. CD47 expression at the cell membrane, as judged by flow cytometry, was markedly reduced within 30 minutes using actin stabilising drugs (Cytochalasin D (5µM): Exp 13.7±5.4% versus T48 0.5±5.7%; Latrunculin A (1µM): Exp 18.9±3.5% versus T48 9.9±5.9%, of the DMSO control), and destabilising drug (Jasplakinolide (1µM): Exp 24.2±1.9% versus T48 -6±1.8%, of the DMSO control), until the basophilic erythroblast stage. In K562 cells, which predominantly express CD47 isoforms 3 and 4, a larger actin dependency is observed (37±14.9% reduction in CD47 with Cytochalasin D versus a DMSO control) suggesting that dependence on actin by CD47 is not isoform specific. In summary, we propose a role for actin in the maintenance of CD47 at the cell surface before and during early erythroid differentiation. We have shown that CD47 isoform 2 is the major isoform present at the cell surface and that this version is initially dependent on the actin cytoskeleton for its membrane stability by an as yet undetermined mechanism. Once band 3 complex assembly initiates at the surface of the basophilic erythroblast (48 hours post-differentiation), CD47 is selectively incorporated via an interaction with protein 4.2, and is preferentially retained whilst the actin cytoskeleton remodels. In addition to explaining how CD47 expression is maintained during the formation of the red cell membrane, this work raises the possibility that the dependence on actin by CD47 for its membrane stability in hematopoietic stem cells may be exploited for the development of therapeutics that render the leukemic cells susceptible to phagocytosis. Disclosures No relevant conflicts of interest to declare.

Changes in the number and volume of fibrillar centres with the inactivation of nucleoli at erythropoiesis

The number and volume of fibrillar centres, the structural components of interphase cell nucleoli on the surface of which rRNA is synthesized, have been studied in differentiating erythroblasts of mouse embryo liver. Complete series of ultrathin sections of erythroblast nuclei have been used at the main stages of differentiation: proerythroblast, basophilic erythroblast, polychromatophilic erythroblast and normoblast. It has been shown that in the active nucleoli of proerythroblasts the number of fibrillar centres is correlated with cell ploidy and exceeds by several-fold the number of nucleolus-organizing regions of chromosomes. The total volumes of fibrillar centres in 2C (0.369 micron 3) and 4C (0.749 micron 3) proerythroblasts are proportional to number of nucleolus-organizing regions. With the maturation of erythroblasts the total number of fibrillar centres declines and in normoblasts it is 3- to 10-fold less than that of the nucleolus-organizing regions. The total volume of fibrillar centres in normoblasts (0.102 micron 3) is threefold smaller than that in proerythroblasts (0.369 micron 3), even though the mean volumes of individual fibrillar centres are significantly higher (0.0042 micron 3 in proerythroblasts and 0.039 in normoblasts). The optical density of fibrillar centres in normoblasts can be higher compared with that of proerythroblasts. It has been suggested that the inactivation of nucleoli at erythropoiesis is accompanied by the fusion of individual fibrillar centres and, possibly, by the compaction of their material.

Half-life and rate of synthesis of globin messenger ribonucleic acid. Determination of half-life of messenger ribonucleic acid and its relative synthetic rate in erythroid cells

The specific radioactivity of mouse globin mRNA in blood reticulocytes was measured after injection of [3H]uridine into anaemic mice up to 60h before collection of reticulocytes. From these data, the decay of the acid-soluble nucleotide pool in the marrow and the relative marrow-cell composition it is possible to build models that allow the cell life-times and half-life of mRNA in the erythroid cells of the marrow to be calculated. Best fit of models to these data favour a model with either one or two cell divisions from the onset of mRNA synthesis. The single-cell-division model has cell times of 20, 13 and 7h respectively for the basophilic erythroblast, polychromatophilic erythroblast and reticulocyte. The two-cell-division model has cell times of 12, 12, 12 and 7h for the basophilic erythroblast 1 and 2, polychromatophilic erythroblast and reticulocyte respectively. Both models have an mRNA half-life of 17h and a constant rate of mRNA synthesis until enucleation at the reticulocyte stage, when synthesis stops. A declining rate of mRNA synthesis can be accommodated in a two-cell-division model, when synthesis halves at each cell division and cell times are essentially the same as above, but mRNA half-life is either 9h in the basophilic and polychromatophilic erythroblasts and 17h in the later cells, or 10h in the basophilic erythroblasts and polychromatophilic erythroblasts and 14.5h in later cells. In all cases it is clear that mRNA synthesis occurs over a time-period of only 30–36h and that mRNA cannot be pre-synthesized in precursor erythroid cells.

Light- and Electron-Microscope Studies on the Spleen of the Newt Triturus Cristatus: The Fine Structure of Erythropoietic Cells

The stages of erythrocyte maturation were identified in spleen smears stained with May-Grünwald-Giemsa. The fine structure of sectioned cells from about the basophilic erythroblast stage onwards was investigated in the electron microscope and serial I-µ sections were examined by microspectrophotometry to ascertain haemoglobin content. The nuclei of basophilic erythroblasts contain large and small blocks of chromatin as well as light-staining zones of unknown composition and ill-defined structure: the nuclear sap contains numerous interchromatin granules, about 400 Å in diameter. During maturation the small blocks of chromatin aggregate and the nuclear light-staining zones tend to disappear, as do the interchromatin granules. Storage lysosomes occur in the basophilic and early polychromatic erythroblasts and during subsequent maturation these lysosomes are probably involved in the degradation of mitochondria. The changes in distribution of ribosomes in cells at the later stages of maturation have been investigated by counting the numbers of single ribosomes and polysomes seen in electron micrographs. During erythropoiesis the ratio of the amount of fibrillar material to the amount of granular material in the nucleolus increases; in the mature erythrocyte the nucleolus consists almost exclusively of fine fibrillar material.