scholarly journals Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation.

1988 ◽  
Vol 107 (2) ◽  
pp. 413-426 ◽  
Author(s):  
M E Lehnert ◽  
H F Lodish
Keyword(s):  
Single Injection ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Cytoskeletal Proteins ◽  
Protein 4.1 ◽  
Stage Of Development ◽  
Alpha Spectrin ◽  
Actin Mrna ◽  
Murine Erythroleukemia ◽  
Differentiation Period

Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.

scholarly journals A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes.

1987 ◽  
Vol 105 (6) ◽  
pp. 3105-3118 ◽  
Author(s):  
V P Patel ◽  
H F Lodish
Keyword(s):  
Suspension Culture ◽  
Erythroid Differentiation ◽  
Protein Band ◽  
Band 3 ◽  
Fibronectin Receptor ◽  
Erythroid Precursor ◽  
Culture Cells ◽  
Fibronectin Matrix ◽  
Normal Differentiation ◽  
Murine Erythroleukemia

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 lysis. In contrast, cells induced by DMSO on fibronectin-coated dishes for 7 d differentiate into enucleating cells, reticulocytes, and erythrocytes. As determined by quantitative immunoblotting, cells induced in suspension culture accumulate approximately 33% of the amount of the major erythroid membrane protein Band 3 present in erythrocyte, whereas cells induced on fibronectin-coated dishes accumulate 80-100% of the amount present in erythrocytes. Both suspension-induced cells and cells induced on fibronectin-coated dishes accumulate approximately 90% of the amount of spectrin and ankyrin present in erythrocytes. As revealed by immunofluorescence microscopy during enucleation of MEL cells, both Band 3 and ankyrin are sequestered in the cytoplasmic fragment of the emerging reticulocyte. Enucleated and later-stage cells detach from the fibronectin matrix, due to the loss of the surface fibronectin receptor; this mimics the normal release of reticulocytes from the matrix of the bone marrow into the blood. Thus a fibronectin matrix provides a permissive microenvironment within which erythroid precursor cells reside, proliferate, migrate, and express their normal differentiation program.

scholarly journals Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells

2010 ◽  
Vol 432 (2) ◽  
pp. 407-416 ◽  
Author(s):  
Wataru Nunomura ◽  
Kengo Kinoshita ◽  
Marilyn Parra ◽  
Philippe Gascard ◽  
Xiuli An ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Erythroid Cells ◽  
Protein 4.1 ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
And Function ◽  
Glycophorin C ◽  
In Erythroid Cells

Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. Although the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high-affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.

scholarly journals miR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis

2021 ◽  
Vol 14 (2) ◽  
pp. 137
Author(s):  
Christos I. Papagiannopoulos ◽  
Nikoleta F. Theodoroula ◽  
Ioannis S. Vizirianakis
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Cultured Cells ◽  
Erythroid Differentiation ◽  
Underlying Mechanism ◽  
Loss Of Function ◽  
Functional Studies ◽  
Differentiated Cells ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

miRNAs constitute a class of non-coding RNA that act as powerful epigenetic regulators in animal and plant cells. In order to identify putative tumor-suppressor miRNAs we profiled the expression of various miRNAs during differentiation of erythroleukemia cells. RNA was purified before and after differentiation induction and subjected to quantitative RT-PCR. The majority of the miRNAs tested were found upregulated in differentiated cells with miR-16-5p showing the most significant increase. Functional studies using gain- and loss-of-function constructs proposed that miR-16-5p has a role in promoting the erythroid differentiation program of murine erythroleukemia (MEL) cells. In order to identify the underlying mechanism of action, we utilized bioinformatic in-silico platforms that incorporate predictions for the genes targeted by miR-16-5p. Interestingly, ribosome constituents, as well as ribosome biogenesis factors, were overrepresented among the miR-16-5p predicted gene targets. Accordingly, biochemical experiments showed that, indeed, miR-16-5p could modulate the levels of independent ribosomal proteins, and the overall ribosomal levels in cultured cells. In conclusion, miR-16-5p is identified as a differentiation-promoting agent in erythroleukemia cells, demonstrating antiproliferative activity, likely as a result of its ability to target the ribosomal machinery and restore any imbalanced activity imposed by the malignancy and the blockade of differentiation.

scholarly journals Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells

Blood ◽  
1979 ◽  
Vol 54 (4) ◽  
pp. 933-939
Author(s):  
R Gambari ◽  
RA Rifkind ◽  
PA Marks
Keyword(s):  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Globin Mrna ◽  
Induced Differentiation ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Mrna Content ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.

scholarly journals Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3934-3941 ◽  
Author(s):  
F Baklouti ◽  
SC Huang ◽  
TK Tang ◽  
J Delaunay ◽  
VT Marchesi ◽  
...  
Keyword(s):  
Erythroid Differentiation ◽  
Mrna Splicing ◽  
Actin Binding ◽  
Splice Acceptor Site ◽  
Erythroid Cell ◽  
Junctional Complex ◽  
Mrna Isoforms ◽  
Exon 2 ◽  
Protein 4.1 ◽  
Alternative Mrna Splicing

Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5′ end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high-molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD “spectrin/actin binding domain” (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5′ end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma.

scholarly journals Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1362-1370 ◽  
Author(s):  
JO Hensold ◽  
G Dubyak ◽  
DE Housman
Keyword(s):  
Transcriptional Activation ◽  
Calcium Ionophore ◽  
Erythroid Differentiation ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cellular Processes ◽  
Cellular Events ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

Abstract Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

scholarly journals Increased rotational mobility and extractability of band 3 from protein 4.2-deficient erythrocyte membranes: evidence of a role for protein 4.2 in strengthening the band 3-cytoskeleton linkage

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2745-2753 ◽  
Author(s):  
AC Rybicki ◽  
RS Schwartz ◽  
EJ Hustedt ◽  
CE Cobb
Keyword(s):  
Correlation Time ◽  
Osmotic Fragility ◽  
Band 3 ◽  
Cytoskeletal Proteins ◽  
Erythrocyte Membranes ◽  
Rotational Mobility ◽  
Direct Role ◽  
Emission Anisotropy ◽  
Protein 4.2 ◽  
Nonionic Detergent

Band 3 (anion-exchange protein 1-[AE1]) is the major integral membrane protein of human erythrocytes and links the membrane to the underlying cytoskeleton via high-affinity binding to ankyrin. It is unclear whether other cytoskeletal proteins participate in strengthening the ankyrin-band 3 linkage, but a putative role for protein 4.2 (P4.2) has been proposed based on the increased osmotic fragility and spherocytic morphology of P4.2-deficient red blood cells (RBCs). The present study was designed to investigate the hypothesis that P4.2 has a direct role in strengthening the band 3-cytoskeleton linkage in human RBCs, by measuring independent features of this interaction in normal and P4.2-deficient RBCs. The features examined were the rotational mobility of band 3 assayed by time-resolved phosphorescence emission anisotropy (TPA), and the extractability of band 3 by octyl-beta-glucoside, the latter being a nonionic detergent that selectively extracts only band 3 that is not anchored to the cytoskeleton. We find that the amplitude of the most rapidly rotating population of band 3 (correlation time, approximately 30 to 60 microseconds) is increased 81% and 67% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts. The amplitude of the intermediate speed rotating population of band 3 (correlation time, approximately 200 to 500 microseconds) is increased 23% and 8% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts, at the expense of the slowly rotating component (correlation time, approximately 2,000 to 3,000 microseconds, amplitude decreased 43% and 39% in P4.2NIPPON and band 3MONTEFIORE, respectively) and immobile component (immobile on this experimental time scale; amplitude decreased 26% and 10% in P4.2NIPPON and band 3MONTEFIORE, respectively) of band 3. These results demonstrate that P4.2 deficiency partially removes band 3 rotational constraints, ie, it increases band 3 rotational mobility. The nonionic detergent octyl-beta-glucoside, which does not disturb band 3-cytoskeleton associations, ie, it extracts only band 3 that is not attached to the cytoskeleton, extracted 30% and 61% more band 3 from P4.2NIPPON and band 3MONTEFIORE ghost membranes, respectively, compared with control ghosts. The octyl-beta-glucoside ghost extracts from both P4.2-deficient phenotypes were enriched in band 3 oligomeric species (tetramers, higher-order oligomers, and aggregates) compared with controls. Since band 3 oligomers selectively associate with the cytoskeleton, these results are consistent with a weakened band 3-cytoskeleton linkage in P4.2-deficient RBC membranes. P4.2 deficiency does not affect band 3 anion transport activity, since uptake of radiolabeled sulfate was similar for control and P4.2-deficient RBCs. Thus, we propose that P4.2 directly participates in strengthening the band 3-cytoskeleton linkage.

Phenotypic plasticity in the intercalated cell: the hensin pathway

1998 ◽  
Vol 275 (2) ◽  
pp. F183-F190 ◽  
Author(s):  
Qais Al-Awqati ◽  
S. Vijayakumar ◽  
C. Hikita ◽  
J. Chen ◽  
J. Takito
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cell Types ◽  
Band 3 ◽  
Cytoskeletal Proteins ◽  
Β Cell ◽  
Biochemical Basis ◽  
Intercalated Cell ◽  
Immortalized Cell

The collecting duct of the renal tubule contains two cell types, one of which, the intercalated cell, is responsible for acidification and alkalinization of urine. These cells exist in a multiplicity of morphological forms, with two extreme types, α and β. The former acidifies the urine by an apical proton-translocating ATPase and a basolateral Cl/HCO3 exchanger, which is an alternately spliced form of band 3. This kidney form of band 3, kAE1, is present in the apical membrane of the β-cell, which has the H+-ATPase on the basolateral membrane. We had suggested previously that metabolic acidosis leads to conversion of β-types to α-types. To study the biochemical basis of this plasticity, we used an immortalized cell line of the β-cell and showed that these cells convert to the α-phenotype when plated at superconfluent density. At high density these cells localize a new protein, which we term “hensin,” to the extracellular matrix, and hensin acts as a molecular switch capable of changing the phenotype of these cells in vitro. Hensin induces new cytoskeletal proteins, makes the cells assume a more columnar shape and retargets kAE1 and the H+-ATPase. These recent studies suggest that the conversion of β- to α-cells, at least in vitro, bears many of the hallmarks of terminal differentiation.

scholarly journals Relationship between globin mRNA accumulation and commitment to terminal cell differentiation in inducer sensitive and resistant erythroleukemia variants

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 302-306
Author(s):  
N Weich ◽  
PA Marks ◽  
RA Rifkind
Keyword(s):  
Cell Differentiation ◽  
Erythroid Differentiation ◽  
Terminal Cell ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Chemical Inducers ◽  
Transformed Cell Line ◽  
Increased Sensitivity ◽  
Differentiation Inducers ◽  
Murine Erythroleukemia

Abstract The relationship between the kinetics of commitment to terminal cell differentiation and the rates of accumulation of globin mRNA has been examined during the induction of erythroid differentiation by polar/apolar chemical inducers in murine erythroleukemia cells (MELC), under conditions of more and less rapid commitment. Two differentiation inducers and three MELC variants have been studied. Hexamethylene bisacetamide (HMBA) initiates more rapid commitment than does dimethylsulfoxide (Me2SO). MELC variant DR10 is resistant to induction by Me2SO and responds sluggishly to HMBA, in comparison with the DS19- Sc9 variant. V3.17, an MELC variant resistant to low concentrations of vincristine, shows increased sensitivity to the inducers and an accelerated rate of commitment to terminal differentiation compared with DS19-Sc9. It is demonstrated that commitment and the actual expression of differentiation, as measured by the accumulation of alpha- , beta maj-, and beta min-globin mRNA, are temporally coordinated functions during induced differentiation of a transformed cell line by exposure to polar/apolar agents.

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