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Immunoblot shows induction of Band 3 protein during MEL cell differentiation. The indicated number of erythrocyte ghosts (lanes 2-7), cells induced in suspension culture (lanes 9 and 10), and on Fn-coated dishes (lanes 13-18), and nninduced cells growing in suspension culture (lane 8) or on Fn-coated dishes (lane 12) were subjected to SDS-PAGE and immunoblotting as described in Materials and Methods. A sample of ~2SI-labeled erythrocyte ghosts was run as molecular weight marker (lane 1). No sample was applied to lane //. U, unattached cells; A, attached cells.
Source publication
Erythroid differentiation of murine erythroleukemia (MEL) cells is far more extensive when the cells are attached to fibronectin-coated dishes than in suspension culture. Cells induced in suspension culture for 4 d become arrested at a late erythroblast stage and do not undergo enucleation. Incubation of cells in suspension beyond 4 d results in ly...
Contexts in source publication
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... ghosts (Fig. 3, lane 9, and Table IV). However, an additional 2 d of incubation resulted in a dramatic de- crease in the cellular content of spectrin (Fig. 3, lane 10, and Table IV). Note that the uneven intensities of spectrin subunits on the immunoblots reflects the preferential reactiv- ity of the anti-spectrin antisera towards ct-spectrin; both r and 13-subunits of spectrin were efficiently transferred from the gel to nitrocellulose sheet, as is shown here by the sample containing iodinated erythrocyte ghosts ( Fig. 2 and 3, lane 1). This was also confirmed by staining the nitrocel- lulose sheet with India ink after the transfer (not shown). Suspension cultured cells also synthesized ankyrin in re- sponse to DMSO treatment. The steady-state level of ankyrin after 4 d of differentiation corresponded to 85 % of the anky- rin present in the erythrocyte ghosts (Fig. 4, lane 9, and Table IV). As with Band 3 and spectrin, the level of ankyrin de- clined sharply when cells were incubated for an additional 2 d in suspension culture (Fig. 4, lane 10, and Table ...
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... observations suggested that the full developmental potential of MEL cells can be induced by a fibronectin ma- trix. To biochemically characterize the differentiation pro- cess, we measured by the Western blotting technique the lev- els of Band 3, spectrin, and ankyrin in cells induced either in suspension culture or on Fn-coated dishes. To quantitate the amounts of these proteins in MEL cells relative to those in erythrocytes and to assess the variabilities in protein trans- fer and antibody reaction, known amounts of mouse erythro- cyte ghosts were analyzed in parallel with the MEL cell sam- pies on the same gel. The amount of ~I-labeled protein A associated with each of the erythrocyte standard protein was proportionate (over a sixfold range) to the amount of erythro- cyte ghosts applied to the gel, shown here for Band 3 (Fig. 2, lanes 2-7), spectrin (Fig. 3, lanes 2-7), and ankyrin (Fig. 4, lanes 2-7). Amounts of Band 3, spectrin, and ankyrin in MEL cells were estimated by the Western blotting technique as described in Materials and Methods. The unat- tached population of cells recovered on days 4, 6, and 7 consists of cells that detached from Fn-coated dishes during 0-4, 4-6, and 6-7 d, respectively. 3 x 105 (for band 3 and spectrin) and 4 x 10 ~ (for ankyrin) MEL cell equiva- lents were subjected to SDS-PAGE and immunoblotting. The relevant bands were excised and the amount of radioactivity was determined with a gamma counter. Values presented are expressed as percentage of total radioactivity de- tected when equivalent number of erythrocyte ghosts were subjected to SDS- PAGE and ...
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... observations suggested that the full developmental potential of MEL cells can be induced by a fibronectin ma- trix. To biochemically characterize the differentiation pro- cess, we measured by the Western blotting technique the lev- els of Band 3, spectrin, and ankyrin in cells induced either in suspension culture or on Fn-coated dishes. To quantitate the amounts of these proteins in MEL cells relative to those in erythrocytes and to assess the variabilities in protein trans- fer and antibody reaction, known amounts of mouse erythro- cyte ghosts were analyzed in parallel with the MEL cell sam- pies on the same gel. The amount of ~I-labeled protein A associated with each of the erythrocyte standard protein was proportionate (over a sixfold range) to the amount of erythro- cyte ghosts applied to the gel, shown here for Band 3 (Fig. 2, lanes 2-7), spectrin (Fig. 3, lanes 2-7), and ankyrin (Fig. 4, lanes 2-7). Amounts of Band 3, spectrin, and ankyrin in MEL cells were estimated by the Western blotting technique as described in Materials and Methods. The unat- tached population of cells recovered on days 4, 6, and 7 consists of cells that detached from Fn-coated dishes during 0-4, 4-6, and 6-7 d, respectively. 3 x 105 (for band 3 and spectrin) and 4 x 10 ~ (for ankyrin) MEL cell equiva- lents were subjected to SDS-PAGE and immunoblotting. The relevant bands were excised and the amount of radioactivity was determined with a gamma counter. Values presented are expressed as percentage of total radioactivity de- tected when equivalent number of erythrocyte ghosts were subjected to SDS- PAGE and ...
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... observations suggested that the full developmental potential of MEL cells can be induced by a fibronectin ma- trix. To biochemically characterize the differentiation pro- cess, we measured by the Western blotting technique the lev- els of Band 3, spectrin, and ankyrin in cells induced either in suspension culture or on Fn-coated dishes. To quantitate the amounts of these proteins in MEL cells relative to those in erythrocytes and to assess the variabilities in protein trans- fer and antibody reaction, known amounts of mouse erythro- cyte ghosts were analyzed in parallel with the MEL cell sam- pies on the same gel. The amount of ~I-labeled protein A associated with each of the erythrocyte standard protein was proportionate (over a sixfold range) to the amount of erythro- cyte ghosts applied to the gel, shown here for Band 3 (Fig. 2, lanes 2-7), spectrin (Fig. 3, lanes 2-7), and ankyrin (Fig. 4, lanes 2-7). Amounts of Band 3, spectrin, and ankyrin in MEL cells were estimated by the Western blotting technique as described in Materials and Methods. The unat- tached population of cells recovered on days 4, 6, and 7 consists of cells that detached from Fn-coated dishes during 0-4, 4-6, and 6-7 d, respectively. 3 x 105 (for band 3 and spectrin) and 4 x 10 ~ (for ankyrin) MEL cell equiva- lents were subjected to SDS-PAGE and immunoblotting. The relevant bands were excised and the amount of radioactivity was determined with a gamma counter. Values presented are expressed as percentage of total radioactivity de- tected when equivalent number of erythrocyte ghosts were subjected to SDS- PAGE and ...
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... enucleation of the normal mammalian erythroblast, the erythrocyte membrane sialoglycoproteins and spectrin are retained by the reticulocyte (17, 42). To localize Band 3 and ankyrin in differentiating cultures of MEL cells on Fn- coated glass slides, cells were fixed with paraformaldehyde and incubated with the appropriate antibody. The bound anti- body was then detected by fluorescence microscopy using rhodamine-conjugated goat anti-rabbit IgG. No fluorescence was detected when cells at any stage of development were in- cubated with either the preimmune IgG plus the secondary antibody or the secondary antibody alone (not shown). Band 3 was barely detectable in uninduced MEL cells (Fig. 5 b), a result consistent with our immunoblotting data (Fig. 2, lane 12, and Table IV). After 4 d of differentiation, cells ex- pressed high levels of Band 3 and it was uniformly distrib- uted throughout the cytoplasm in both the fibronectin-at- tached ( Fig. 5 c) and unattached population of cells (not shown). Some cells exhibited punctate clusters of Band 3. After 7 d of differentiation, most of the Band 3 was sequest- ered in the cytoplasmic fragment of the emerging reticulo- cyte (Fig. 5 ...
Citations
... We obtained MEL (Murine Erythroleukemia) cells from Dr. John Crispino. MEL cells can be propagated in culture indefinitely and exhibit many characteristics of erythroblasts (Singer et al. 1974;Antoniou 1991), including enucleation (Volloch and Housman 1982;Patel and Lodish 1987). In this study, we investigated the role of the licensing ORC4 protein in erythroblast enucleation by using MEL cells and demonstrated that ORC4 is required MEL cell enucleation. ...
The Origin Replication Complex subunit 4 (ORC4) is one in six subunits of the Origin Replication Complexes (ORCs) which is essential for initiating licensing at DNA replication origins and recruiting adaptor molecules necessary for various cellular processes. Previously, we reported that ORC4 also plays a vital role in polar body extrusion (PBE) during oogenesis in which half the chromosomes are extruded from the oocyte. We hypothesized that ORC4 might play a broader role in chromatin elimination. We tested its role in enucleation during the development of erythrocytes. Murine erythroleukemia (MEL) cells can be propagated in culture indefinitely and can be induced to enucleate their DNA by treatment with Vacuolin-1, thereby mimicking normal erythrocyte enucleation. We found that ORC4 appeared around the nuclei of the MEL cells with Vacuolin-1 treatment, gradually increasing in thickness before enucleation. We then tested whether ORC4 was required for MEL enucleation by down regulating ORC4 with siRNA-ORC4 during Vacuolin-1 treatment and found that this prevented MEL enucleation. These data are consistent with the model that ORC4 is required for erythroblast enucleation just as it is for oocyte PBE. They suggest a new model in which ORC4 expression is a marker for the initiation to the enucleation pathway.
... All the aforementioned data provide a molecular conformation of the phenotype described earlier in primary and transplanted ko cells and suggest that ko erythroid cells are intrinsically unable to expediently assemble under stress conditions the complete erythroid differentiation profile required for optimal response. Discussion a5 integrin and fibronectin in erythropoiesis It was first recognized more than 30 years ago that interactions of erythroid cells with fibronectin (Fn) in hematopoietic tissues are important for the proliferation and differentiation of erythroid cells [15]. This interaction was thought to be exercised mainly by a5 integrin in erythroid cells having Fn as the exclusive ligand. ...
We have previously studied the role of β1 integrin and some of its different alpha partners relevant to Erythropoiesis. Although clear and consistent answers regarding the role of α4β1 (VLA-4) were evident, the role of its companion integrin α5β1 (VLA-5) was clouded by inconsistent outcomes in all prior publications. Furthermore, the functional consequences of integrin deficiencies only in microenvironmental cells (ME) supporting erythroid cell expansion and maturation post stress have never been explored. In the present studies we have created several additional mouse models aiming to address unanswered questions regarding functional consequences of single or combined integrin deficiencies in erythroid cells or only in ME supporting cells. Our novel and expansive data solidified the intrinsic requirement of both α4 and α5 integrins in erythroid cells for their proliferative expansion and maturation in response to stress; α5 integrin alone, deleted either early in all hematopoietic cells or only in erythroid cells has only a redundant role in proliferative expansion and is dispensable for erythroid maturation. By contrast, α4 integrin, on its own, exerts a dominant effect on timely and optimal erythroid maturation. Deficiency of both α4 and α5 integrins in ME cells including macrophages does not negatively influence stress response by normal erythroid cells, in great contrast to the effect of ME cells deficient in all β1 integrins. Collectively the present data offer a deeper insight on the coordination of different β1 integrin functional activities in erythroid cells or in ME cells for optimal erythroid stress response.
... When treated with inducers of differentiation, MEL cells can complete the differentiation program and reach the reticulocyte stage. Under specific conditions, when grown in the presence of a fibronectin matrix, a large proportion of MEL cells will complete enucleation (Patel & Lodish, 1987). MEL-R cells, however, are unable to grow on fibronectin-coated plates and cannot complete enucleation loss (Fernandez-Nestosa, 2007). ...
Wiskott-Aldrich syndrome (WAS) is a recessive X-linked inmmunodeficiency caused by loss-of-function mutations in the gene encoding the WAS protein (WASp). WASp plays an important role in the polymerization of the actin cytoskeleton in hematopoietic cells through activation of the Arp2/3 complex. In a previous study, we found that actin cytoskeleton proteins, including WASp, were silenced in murine erythroleukemia cells defective in differentiation. Here, we designed a CRISPR/Cas9 strategy to delete a 9.5-kb genomic region encompassing the Was gene in the X chromosome of murine erythroleukemia (MEL) cells. We show that Was -deficient MEL cells have a poor organization of the actin cytoskeleton that can be recovered by restoring Was expression. We found that whereas the total amount of actin protein was similar between wild-type and Was knockout MEL cells, the latter exhibited an altered ratio of monomeric G-actin to polymeric F-actin. We also demonstrate that Was overexpression can mediate the activation of Bruton’s tyrosine kinase. Overall, these findings support the role of WASp as a key regulator of F-actin in erythroid cells.
... It is therefore possible that adhesion to the extracellular environment, e.g. Emp (erythroblasts macrophage protein) dependent adhesion between erythroblasts and macrophages [32,33], or adhesion between erythroblasts and fibronectin [34], or possibly adhesion amongst erythroblasts [35], could provide the necessary cues to control asymmetric positioning of the cytoskeleton prior and during enucleation, rather than cellintrinsic asymmetry. The observation that erythroid enucleation can occur in vitro in the absence of many of these microenvironmental cues though also suggests that other non-deterministic and stochastic mechanisms could be utilized to generate these asymmetries. ...
Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation.
... Thus, it is not surprising that various functional impairments described in either macrophages or Ebs are mainly manifested under stress. Molecules providing adhesive contact between macrophages and Ebs (alpha4 integrin/VCAM-1, 8,19,20 ICAM4/alphav/alpha4 integrins, 10,11,21,22 CD163, 23 palladin, 12 as well as extracellular matrix proteins, fibronectin, 24,25 and laminin), [26][27][28] exert regulatory roles mostly under stress by influencing growth, differentiation, and adhesion/migration of erythroid cells. In addition to direct contact, soluble factors secreted by either Ebs or macrophages influence stress erythropoiesis. ...
... In response to Epo, Ebs release Gas6 (important for cell survival) by boosting Epo receptor signaling and by enhancing adhesion to fibronectin through very-late-activation antigen 4 activation. 24,25 Data in Gas6 2/2 mice convincingly show that Gas6 regulates the response to acute anemia in both the spleen and BM. 29 Apart from the erythroid direct effects, Gas6 exerts a paracrine effect by dampening the release of erythroidinhibitory factors by macrophages. Further, erythroid cells, in addition to Gas6, secrete two angiogenic factors, VEGF-A and PlGF. ...
Key Points
Significant expansion only of native splenic macrophages that are F4/80+/Cd11blo occurs in both post-Epo and post–hemolysis-induced stress. VCAM-1−/− mice, like Spi-C−/−, mice have significantly decreased macrophages but did not have a compromised E-stress response.
... Currently, genes polymorphism analyses have seen increased usage in the study of the molecular mechanism of calcium oxalate stones, and has been applied to discover the relationship between the occurrence of calcium oxalate stones and polymorphisms in the γ-hydroxybenzyl thiocyanide glutamic acid, vitamin D receptor, and calcitonin receptor genes, among others (Bid et al., 2005;Gao et al., 2007;Metin et al., 2009). Patel andLodish (1987) mapped the Fn gene to chromosome 2q34-36, and reported the presence of 50 exons. A majority of these exons corresponded to the repeat sequences of peptide chains. ...
Here, we have investigated the correlation between calcium oxalate stone formation and Fn gene polymorphisms in urinary calculi patients among the Uighur population (Xinjiang region). In this case control study, genomic DNA extracted from the peripheral blood of 129 patients with calcium oxalate stones (patient group) and 94 normal people (control group) was used to genotype polymorphisms in the rs6725958, rs10202709, and rs35343655 sites of the Fn gene by polymerase chain reaction-restriction fragment length polymorphism. Subsequently, the association between different genotypes and susceptibility to calcium oxalate stone formation was compared among the patient and control groups. Single nucleotide polymorphisms (SNPs) were detected in the rs6725958, rs10202709, and rs35343655 sites of the Fn gene among the patient and control groups. The genotype distributions of the three loci complied with the Hardy-Weinberg equilibrium. The results of allele frequencies of the patient/control group for polymorphisms in the rs6725958 site of the Fn gene were C = 179 (69.92%)/119 (63.30%) and A = 77 (30.08%)/69 (36.70%), in the rs10202709 site were C = 245 (95.70%)/176 (93.63%) and T = 11 (4.30%)/12 (6.38%), and in the rs35343655 site of the Fn gene were A = 139 (54.30%)/87 (46.28%) and G = 117 (45.70%)/101 (53.72%). We observed no significant differences between the three SNPs and development of calcium oxalate stones. Polymorphisms in rs6725958, rs10202709, and rs35343655 of the Fn gene had no obvious effect on the susceptibility to the development of calcium oxalate stones in the Uighur population, residing in the Xinjiang region of China.
... Second, it is important to use low endotoxin BSA in Epo medium since it is critical for enucleation, although the mechanism is not clear. Third, although fibronectin was reported to be important for erythropoiesis 15 , we found that the use of fibronectin coated plate was optional. It does not affect cell proliferation, differentiation, or enucleation in our system. ...
... To date several membrane and cytoskeletal proteins are known to differentially sort to the reticulocyte in humans and mice. The membrane proteins band 3 [47,48], GPA [47], GPC [47] and RhAG [47] are reported to be restricted to the reticulocyte in murine cells. Alongside the spectrin cytoskeleton [45,46], microtubules [40,46], myosin [40] and actin also were restricted to the reticulocyte in mouse cells. ...
Enucleation represents the critical stage during red blood cell development when the nucleus is extruded from an orthochromatic erythroblast in order to generate a nascent immature reticulocyte. Extrusion of the nucleus results in loss of a proportion of the erythroblast plasma membrane, which surrounds the nucleus, the bulk of the endoplasmic reticulum and a small region of cytoplasm. For this reason, enucleation provides an important point in erythroblast differentiation at which proteins not required for the function of the erythrocyte can be lost, whilst those that are important for the structure-function properties of the mature erythrocyte must be efficiently retained in the reticulocyte plasma membrane. Disturbances in protein distribution during enucleation are envisaged to occur during human diseases such as hereditary spherocytosis. This article will discuss our current understanding of erythroblast enucleation in the context of retention and loss of proteins that display antigenic blood group sites, that are known to exist within multiprotein complexes within the erythrocyte membrane.
... Other ECM studies have shown that each ECM protein influences cellular growth through a unique sequence [Adams and Watt, 1993;Huang and Ingber, 1999;Muller et al., 2012]. For example, various types of fibronectin play a role in cell binding, cell adhesion and cell motility as well as cell division [Patel and Lodish, 1987;Rosso et al., 2004]. Laminin alone or conjugated with other ECMs also improves cell adhesion and survival rate [Klein et al., 1988;von der Mark and Ocalan, 1989]. ...
We present the physical and biochemical effects of extracellular matrixes (ECMs) on HL-1 cardiomyocytes. ECMs play major roles in cell growth, adhesion and the maintenance of native cell functions. We investigated the effects of 6 different cell culture systems: 5 different ECM-treated surfaces (fibronectin, laminin, collagen I, gelatin and a gelatin/fibronectin mixture) and 1 nontreated surface. Surface morphology was scanned and analyzed using atomic force microscopy in order to investigate the physical effects of ECMs. The attachment, growth, viability, proliferation and phenotype of the cells were analyzed using phase-contrast microscopy and immunocytochemistry to elucidate the biochemical effects of ECMs. Our study provides basic information for understanding cell-ECM interactions and should be utilized in future cardiac cell research and tissue engineering. © 2014 S. Karger AG, Basel.
... Integrins such as beta-1 integrin (fibronectin receptor, CD29) function as receptors for the ECM and are important effectors for adhesion, differentiation, and migration of HSCs (Hynes and Yamada 1982;Humphries et al., 1989;Hirsch et al., 1996;Frisch and Ruoslahti 1997;Sugiyama et al., 2011a). Integrin heterodimers and the ECM are thought to interact and function as a homing mechanism to enable HSCs and HPCs to reside in the FL (Patel and Lodish, 1987;Long and Dixit, 1990;Strobel et al., 1997;Sugiyama et al., 2011a). Cells are able to interpret cytokine signaling in particular contexts due to the proteins produced in different cell kinds in the ECM (Taipale and Keski-Oja, 1997;Sugiyama et al., 2011a). ...
The fetal liver (FL) is an important structure in expansion and differentiation of hematopoietic stem cells (HSC), but despite this little is known about the exact mechanisms in which FL hematopoiesis takes place. Primitive hematopoiesis gives way to definitive hematopoiesis at 12.5 dpc in mice and the process is regulated by a number of intrinsic and extrinsic factors. Intrinsic regulations are intracellular processes that have been reported to be important in the initiation of definitive hematopoiesis. Several structures are involved with extrinsic regulations of hematopoiesis within the FL, including hepatoblasts and liver sinusoidal endothelial cells (LSEC). Hepatoblasts and endothelial cells comprise separate niches involved in the extrinsic regulation of hematopoiesis. Studies have shown that co-cultures with fetal liver stromal cells can promote the expansion of erythroid cells, although the way in which stromal cells do this is still unknown. Understanding the mechanisms in which hematopoiesis is regulated in the FL could lead to the production of novel therapies involving the safe and reliable transplantation of HSCs to patients with blood and bone marrow complications. This review aims to summarize the current state of knowledge about the regulation of hematopoiesis specifically within the FL.
