Lamins regulate cell trafficking and lineage maturation of adult human hematopoietic cells.

Hematopoietic stem and progenitor cells, as well as nucleated erythroblasts and megakaryocytes, reside preferentially in adult marrow microenvironments whereas other blood cells readily cross the endothelial barrier into the circulation. Because the nucleus is the largest organelle in blood cells, we hypothesized that (i) cell sorting across microporous barriers is regulated by nuclear deformability as controlled by lamin-A and -B, and (ii) lamin levels directly modulate hematopoietic programs. Mass spectrometry-calibrated intracellular flow cytometry indeed reveals a lamin expression map that partitions human blood lineages between marrow and circulating compartments (P = 0.00006). B-type lamins are highly variable and predominate only in CD34(+) cells, but migration through micropores and nuclear flexibility in micropipette aspiration both appear limited by lamin-A:B stoichiometry across hematopoietic lineages. Differentiation is also modulated by overexpression or knockdown of lamins as well as retinoic acid addition, which regulates lamin-A transcription. In particular, erythroid differentiation is promoted by high lamin-A and low lamin-B1 expression whereas megakaryocytes of high ploidy are inhibited by lamin suppression. Lamins thus contribute to both trafficking and differentiation.

Tetraspanins CD81 and CD82 facilitate α4ß1-mediated adhesion of human erythroblasts to vascular cell adhesion molecule-1.

The proliferation and terminal differentiation of erythroid progenitors occurs in human bone marrow within erythroblastic islands, specialised structures consisting of a central macrophage surrounded by developing erythroid cells. Many cell-cell and cell-matrix adhesive interactions maintain and regulate the co-ordinated daily production of reticulocytes. Erythroid cells express only one integrin, α4ß1, throughout differentiation, and its interactions with both macrophage Vascular Cell Adhesion Molecule-1 and with extracellular matrix fibronectin are critical for erythropoiesis. We observed that proerythroblasts expressed a broad tetraspanin phenotype, and investigated whether any tetraspanin could modulate integrin function. A specific association between α4ß1 and CD81, CD82 and CD151 was demonstrated by confocal microscopy and co-immune precipitation. We observed that antibodies to CD81 and CD82 augmented adhesion of proerythroblasts to Vascular Cell Adhesion Molecule-1 but not to the fibronectin spliceoforms FnIII12-IIICS-15 and FnIII12-15. In contrast, different anti-CD151 antibodies augmented or inhibited adhesion of proerythroblasts to Vascular Cell Adhesion Molecule-1 and the fibronectin spliceoform FnIII12-IIICS-15 but not to FnIII12-15. These results strongly suggest that tetraspanins have a functional role in terminal erythropoiesis by modulating interactions of erythroblast α4ß1 with both macrophages and extracellular matrix.

Membrane remodeling during reticulocyte maturation.

The transition of reticulocytes into erythrocytes is accompanied by extensive changes in the structure and properties of the plasma membrane. These changes include an increase in shear resistance, loss of surface area, and acquisition of a biconcave shape. The processes by which these changes are effected have remained largely undefined. Here we examine how the expression of 30 distinct membrane proteins and their interactions change during murine reticulocyte maturation. We show that tubulin and cytosolic actin are lost, whereas the membrane content of myosin, tropomyosin, intercellular adhesion molecule-4, glucose transporter-4, Na-K-ATPase, sodium/hydrogen exchanger 1, glycophorin A, CD47, Duffy, and Kell is reduced. The degradation of tubulin and actin is, at least in part, through the ubiquitin-proteasome degradation pathway. In regard to the protein-protein interactions, the formation of membrane-associated spectrin tetramers from dimers is unperturbed, whereas the interactions responsible for the formation of the membrane-skeletal junctions are weaker in reticulocytes, as is the attachment of transmembrane proteins to these structures. This weakness, in part, results from the elevated phosphorylation of 4.1R in reticulocytes, which leads to a decrease in shear resistance by reducing its interaction with spectrin and actin. These observations begin to unravel the mechanistic basis of crucial changes accompanying reticulocyte maturation.

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

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.

Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16.

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

Identification of critical amino-acid residues on the erythroid intercellular adhesion molecule-4 (ICAM-4) mediating adhesion to alpha V integrins.

Intercellular adhesion molecule-4 (ICAM-4, syn. LW glycoprotein) interacts with the integrins alpha(L)beta(2), alpha(M)beta(2), A(4)beta(1), the alpha(V) family, and alpha(IIb)beta(3). Systematic mutagenesis of surface-exposed residues conserved between human and murine ICAM-4 defined 12 single amino-acid changes that affect the interaction of ICAM-4 with alpha(V) integrins. Mutation of 10 of these residues, 8 of which are spatially close on the surface of the molecule, led to a reduction in adhesion. Moreover, peptides corresponding to regions of ICAM-4 involved in its interaction with alpha(V) integrins inhibited these interactions. The other 2 mutations increased the extent of interaction of ICAM-4 with alpha(V) integrins. These mutations appear to prevent glycosylation of N160, suggesting that changes in glycosylation may modulate ICAM-4-alpha(V) integrin interactions. The region of ICAM-4 identified as the binding site for alpha(V) integrins is adjacent to the binding sites for alpha(L)beta(2) and alpha(M)beta(2). Selective binding of ICAM-4 to different integrins may be important for a variety of normal red cell functions and also relevant to the pathology of thrombotic disorders and vasoocclusive events in sickle cell disease. Our findings suggest the feasibility of developing selective inhibitors of ICAM-4-integrin adhesion of therapeutic value in these diseases.

Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity.

During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating protein-sorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrin-based skeleton.

Distinct distribution of specific members of protein 4.1 gene family in the mouse nephron.

BACKGROUND: Protein 4.1 is an adapter protein that links the actin cytoskeleton to various transmembrane proteins. These 4.1 proteins are encoded by four homologous genes, 4.1R, 4.1G, 4.1N, and 4.1B, which undergo complex alternative splicing. Here we performed a detailed characterization of the expression of specific 4.1 proteins in the mouse nephron. METHODS: Distribution of renal 4.1 proteins was investigated by staining of paraformaldehyde-fixed mouse kidney sections with antibodies highly specific for each 4.1 protein. Major 4.1 splice forms, amplified from mouse kidney marathon cDNA, were expressed in transfected COS-7 cells in order to assign species of known exon composition to proteins detected in kidney. RESULTS: A 105 kD 4.1R splice form, initiating at ATG-2 translation initiation site and lacking exon 16, but including exon 17B, was restricted to thick ascending limb of Henle's loop. A 95 kD 4.1N splice form, lacking exons 15 and 17D, was expressed in either descending or ascending thin limb of Henle's loop, distal convoluted tubule, and all regions of the collecting duct system. A major 108 kD 4.1B splice form, initiating at a newly characterized ATG translation initiation site, and lacking exons 15, 17B, and 21, was present only in Bowman's capsule and proximal convoluted tubule (PCT). There was no expression of 4.1G in kidney. CONCLUSION: Distinct distribution of 4.1 proteins along the nephron suggests their involvement in targeting of selected transmembrane proteins in kidney epithelium and, therefore, in regulation of specific kidney functions.

A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane.

We have studied the membrane proteins of band 3 anion exchanger (AE1)-deficient mouse and human red blood cells. It has been shown previously that proteins of the band 3 complex are reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3(-/-) RBCs differed from the human band 3-deficient RBCs in that they retained CD47. Aquaporin 1 was reduced, and its glycosylation was altered in mouse and human band 3-deficient RBCs. Proteins of the glycophorin C complex, and other proteins with independent cytoskeletal interactions, were present in normal or increased amounts. To obtain direct evidence for the association of the band 3 and the Rh protein complexes in the RBC, we examined whether Rh complex proteins were coimmunoprecipitated with band 3 from membranes. RhAG and Rh were found to be efficiently coimmunoprecipitated with band 3 from deoxycholate-solubilized membranes. Results suggest that band 3 forms the core of a macrocomplex of integral and peripheral RBC membrane proteins. The presence of these proteins in a single structural macrocomplex makes it likely that they have linked functional or regulatory roles. We speculate that this macrocomplex may function as an integrated CO(2)/O(2) gas exchange unit (metabolon) in the erythrocyte.