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

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 αß-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the ß-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 [(^35)S]methionine, and immunoprecipitation of ankyrin and α- and ß- 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 ß-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of α-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of α- to ß-spectrin. Ankyrin and ß-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 α-spectrin enters the cytoskeletal fraction with similar kinetics to ß-spectrin and ankyrin, and in amounts equimolar to ß-spectrin, the amount of cytoskeletal α-spectrin does not exceed the amount of soluble α-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and α- and ß-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 α- and ß-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

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

Tissue-specific Expression of Distinct Spectrin and Ankyrin Transcripts in Erythroid and Nonerythroid Cells

cDNA probes for three components of the erythroid membrane skeleton, α spectrin, β spectrin, and ankyrin, were obtained by using monospecific antibodies to screen a λgt11 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 β spectrin and ankyrin, while α 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 β spectrin and ankyrin genes underlie the formation of distinct membrane skeletons in erythroid and muscle cells

A plasmid-based system for expressing small interfering RNA libraries in mammalian cells

BACKGROUND: RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21–25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence. RESULTS: To circumvent the limitation of hairpin siRNA vectors we have developed a convergent opposing siRNA expression system called pHippy. We have generated pHippy vectors or expression cassettes that knock down the expression of both reporter and endogenous genes. As a proof of principle that pHippy can be used to generate random siRNA libraries, we generated a small siRNA library against PGL3 luciferase and demonstrated that we could recover functional siRNAs that knock down PGL3 luciferase. CONCLUSIONS: siRNA is a powerful tool to study gene function. We have developed a new vector with opposing convergent promoters for the expression of siRNAs, which can be used to knock down endogenous genes in a high throughput manner or to perform functional screening with random or cDNA-derived siRNA libraries

When Wnts antagonize Wnts

Secreted Wnt ligands appear to activate a variety of signaling pathways. Two papers in this issue now present genetic evidence that “noncanonical” Wnt signaling inhibits the “canonical” Wnt/β-catenin pathway. Westfall et al. (2003a) show that zebrafish embryos lacking maternal Wnt-5 function are dorsalized due to ectopic activation of β-catenin, whereas Topol et al. (2003) report that chondrogenesis in the distal mouse limb bud depends on inhibition of Wnt/β-catenin signaling by a paralogue of Wnt-5. These studies present the first genetic confirmation of the previous hypothesis that vertebrate Wnt signaling pathways can act in an antagonistic manner

Stromelysin-1 and mesothelin are differentially regulated by Wnt-5a and Wnt-1 in C57mg mouse mammary epithelial cells

BACKGROUND: The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca(2+ )pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca(2+ )pathway and whether it regulates gene expression. RESULTS: To test the hypothesis that a specific cell line can respond to distinct Wnts with different patterns of gene expression, we over-expressed Wnt-5a and Rfz-2 in C57mg mammary epithelial cells and compared this cell line to C57mg cells over-expressing Wnt-1. These Wnts were chosen since previous studies suggest that C57mg cells respond differently to these Wnts, and since these Wnts can activate different signaling pathways in other systems. Using DNA microarray analysis, we identified several genes that are regulated by Wnt-5a and Rfz-2 as well as by Wnt-1. We then focused on two genes previously linked to various cancers, mesothelin and stromelysin-1, which are respectively up-regulated by Wnt-1 and Wnt-5a in C57mg cells. CONCLUSION: Different Wnts have distinct effects on gene expression in a single cell line