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R T Moon

Publications and source records attributed to R T Moon.

122 records · Page 7Linked to original sources
PubMed1985

Tissue-specific expression of distinct spectrin and ankyrin transcripts in erythroid and nonerythroid cells.

cDNA probes for three components of the erythroid membrane skeleton, alpha spectrin, beta spectrin, and ankyrin, were obtained by using monospecific antibodies to screen a lambda gt11 expression vector library containing cDNA prepared from chicken erythroid poly(A)+ RNA. Each cDNA appears to hybridize to one gene type in the chicken genome. Qualitatively distinct RNA species in myogenic and erythroid cells are detected for beta spectrin and ankyrin, while alpha spectrin exists as a single species of transcript in all tissues examined. This tissue-specific expression of RNAs is regulated quantitatively during myogenesis in vitro, since all three accumulate only upon myoblast fusion. Furthermore, RNAs for two of the three genes do not accumulate to detectable levels in chicken embryo fibroblasts, demonstrating that their accumulation can be noncoordinate. These observations suggest that independent gene regulation and tissue-specific production of heterogeneous transcripts from the beta spectrin and ankyrin genes underlie the formation of distinct membrane skeletons in erythroid and muscle cells.

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PubMed1985

Anion transporter: highly cell-type-specific expression of distinct polypeptides and transcripts in erythroid and nonerythroid cells.

Affinity-purified antibodies and cDNA probes specific for the chicken erythrocyte anion transporter (also referred to as band 3) have been used to demonstrate that this protein is expressed in a highly cell-type-specific manner in the avian kidney. Indirect immunofluorescence analysis indicates that this polypeptide is present in only a small subset of total kidney cells and is predominantly localized to the proximal convoluted tubule of this organ. Chicken erythrocytes synthesize and accumulate two structurally and serologically related band 3 polypeptides. The polypeptide that accumulates in kidney membranes has an apparent molecular weight greater than either of its erythroid counterparts. This diversity is also reflected at the RNA level, as the single band 3 mRNA species detected during various stages of erythroid development is distinct in size from that found in kidney cells. Genomic DNA blot analysis suggests that both the erythroid and kidney band 3 RNAs arise from a single gene. Furthermore, of the adult tissues we have examined that are known to express ankyrin and spectrin polypeptides, only kidney accumulates detectable levels of the band 3 mRNA and polypeptide. These observations suggest that a subset of kidney cells use an anion transport mechanism analogous to that of erythrocytes and that band 3 is expressed in a noncoordinate manner with other components of the erythroid membrane skeleton in nonerythroid cells.

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PubMed1984

Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin.

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.

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PubMed1983

The cytoskeletal framework of sea urchin eggs and embryos: developmental changes in the association of messenger RNA.

Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.

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PubMed1983

Synthesis and assembly of spectrin during avian erythropoiesis: stoichiometric assembly but unequal synthesis of alpha and beta spectrin.

The synthesis and assembly of spectrin was investigated in erythroid cells during chicken embryo development. Immunoprecipitation of Triton X-100-soluble and -insoluble cytoskeletal fractions with alpha- and beta-spectrin antisera show that, at steady state, alpha and beta spectrin are present in stoichiometric amounts, and exclusively, in the cytoskeleton. However, pulse labeling of cells and in vitro translation of total erythroid cell RNA reveal that alpha spectrin is synthesized in a two to three fold excess over beta spectrin. Pulse-chase experiments show that newly synthesized alpha and beta spectrin are present in both the cytoskeletal and soluble fractions, and that stoichiometric amounts are stably assembled in the cytoskeleton. On the other hand, there is a severalfold excess of alpha relative to beta spectrin in the soluble fraction, both of which turn over with a half-life of 50 min. In cells from 4 day old embryos, more than 80% of the newly synthesized beta spectrin, but only 10% of the alpha spectrin, are present in the cytoskeleton. Thus, early in development, the association of alpha and beta spectrin with the membrane-cytoskeleton may be rate-limited by the amount of beta spectrin synthesized. Later on in erythroid development, progressively lesser proportions of newly synthesized beta spectrin are present in the cytoskeleton, suggesting that during development, the rate of association of beta spectrin with the membrane-cytoskeleton becomes limited by some other membrane-cytoskeletal component.

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PubMed1983

Synthesis and post-translational assembly of intermediate filaments in avian erythroid cells: vimentin assembly limits the rate of synemin assembly.

The assembly of vimentin intermediate filaments and the high molecular weight filament crosslinking protein, synemin, was studied in erythroid cells from 10-day chicken embryos. Pulse labeling studies show that newly synthesized vimentin is present both in a Triton X-100-insoluble form and in a soluble form. The incorporation of labeled vimentin into the insoluble fraction increases linearly with time, while the soluble pool of labeled vimentin saturates quickly. In contrast, synemin accumulates rapidly in the Triton X-100-soluble fraction and begins to accumulate in the insoluble fraction only after a considerable lag of time. Pulse-chase studies reveal that the detergent-soluble pools of both vimentin and synemin contain precursors for their post-translational assembly into detergent-insoluble filaments and that the half-life of soluble synemin is about twice as long as that of soluble vimentin. Immunoprecipitation of solubilized filaments with synemin antiserum precipitates vimentin with synemin. On the other hand, soluble vimentin does not coimmunoprecipitate with soluble synemin. These results suggest that, in the assembly of vimentin and synemin into intermediate filaments, vimentin filament elongation generates synemin binding sites, and thus the rate of vimentin filament elongation limits the rate of synemin assembly.

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PubMed1983

Canavanine inhibits vimentin assembly but not its synthesis in chicken embryo erythroid cells.

In chicken embryo erythroid cells, newly synthesized vimentin first enters a Triton X-100 (TX-100)-soluble pool and subsequently assembles posttranslationally into TX-100-insoluble vimentin filaments (Blikstad I., and E. Lazarides, J. Cell Biol., 96:1803-1808). Here we show that incubation of chicken embryo erythroid cells in a medium in which arginine has been substituted by its amino acid analogue, canavanine, results in the inhibition of the posttranslational assembly of vimentin into the TX-100-insoluble filaments. Immunoprecipitation and subsequent SDS gel electrophoresis showed that the synthesis of canavanine-vimentin is not inhibited and that it accumulates in the TX-100-soluble compartment. Pulse-chase experiments with [35S]methionine demonstrated that while arginine-vimentin can be rapidly chased from the soluble to the cytoskeletal fraction, canavanine-vimentin remains in the soluble fraction, where it turns over. The effect of canavanine on the assembly of vimentin did not prevent the assembly of arginine-vimentin, as cells labeled with [35S]methionine first in the presence of canavanine and then in the presence of arginine contained labeled canavanine-vimentin only in the soluble fraction, and arginine-vimentin in both the soluble and cytoskeletal fractions. These results suggest that arginine residues play an essential role in the assembly of vimentin in vivo.

Animals↗
PubMed1983

Poly(A)-containing messenger ribonucleoprotein complexes from sea urchin eggs and embryos: polypeptides associated with native and UV-crosslinked mRNPs.

Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus. Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm3, and peak buoyant densities in Cs2SO4 in 1.42 g/cm3 after fixation with glutaraldehyde and 1.46 g/cm3 without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5-10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embryonic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.

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PubMed1980

Polypeptides of nonpolyribosomal messenger ribonucleoprotein complexes of sea urchin eggs.

RNA competent in directing protein synthesis is sequestered in unfertilized sea urchin eggs as translationally quiescent, nonpolyribosomal, messenger ribonucleoprotein complexes (mRNPs). Following fertilization, these mRNPs are derepressed and actively translated, presumably due to changes in the mRNA-associated proteins and their interaction with the mRNA. We have isolated poly(A)-containing egg mRNPs free of contaminating monoribosomes and ribosomal subunits by chromatography on oligo(dT)-cellulose and identified their constituent proteins. Egg mRNPs isolated by using near physiological ionic conditions have 15-20 major proteins, most of which are in the molecular weight range of 40 000-100 000, and approximately 15-23 minor proteins in the 22 000-190 000 molecular range. The association of the proteins with poly(A)-containing mRNA is indicated by their greatly reduced retention on oligo(dT)-cellulose after pretreatment of the crude mRNP fraction with saturating amounts of poly(uridylic acid). Three of the proteins present in poly(A)-containing mRNPs from eggs, with molecular weights of 48 000, 67 000, and 140 000, were not detected in poly(A)-containing mRNPs derived from polyribosomes of hatched blastula-stage embryos. In addition, stoichiometric differences were found between some of the proteins associated with the two types of mRNP. The potential regulatory role of these proteins is discussed.

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PubMedDate not supplied

beta-Spectrin limits alpha-spectrin assembly on membranes following synthesis in a chicken erythroid cell lysate.

Spectrin, the major protein of the subcortical actin network in erythrocytes, contains two non-identical subunits, alpha and beta (refs 1, 2). Spectrin is indirectly associated with the transmembrane anion transporter through the binding of beta-spectrin to an extrinsic protein, ankyrin. In chicken embryo erythroid cells, alpha-spectrin is synthesized in a threefold excess relative to beta-spectrin, although the two subunits are assembled in equimolar amounts. To investigate the regulation of assembly of equimolar amounts of spectrin, an in vitro system from chicken embryo erythroid cells has now been developed where synthesis and assembly of spectrin can be uncoupled and studied separately. Following the in vitro translation of threefold more alpha- than beta-spectrin, 95% of the beta-spectrin and equimolar amounts of alpha-spectrin bind post-translationally to spectrin-depleted rabbit red blood cell membranes, and the excess alpha-spectrin remains unbound. This alpha-spectrin cannot bind spectrin-depleted plasma membranes subsequently added to the lysate. The assembly of alpha-spectrin is, therefore, limited by the availability of beta-spectrin, and both subunits assemble post-translationally.

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