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

Upstream Watershed Activities in Iowa and Our Environment

Watershed activities in Iowa have had considerable impact on the welfare and environment of our people. Reductions in floodwater, sediment, and erosion damages to lands have been the principal benefits. Preservation and enhancement of the environment is recognized as a by-product for better living for both rural and urban residents. The Flood Control Act of June 22, 1936, a Congressional appropriation in 1953 of 5 million dollars for Pilot Watershed Projects, and the Watershed Protection and Flood Prevention Act of 1954 (Public Law 566) have been the principal means of initiating watershed projects in Iowa during the past 25 years. The Little Sioux Watershed Project in northwest Iowa was developed under the Flood Control Act of 1936. Three pilot projects were completed, and 35 watershed projects have been authorized for construction under Public Law 566

Long-Wavelength Excesses in Two Highly Obscured High-Mass X-Ray Binaries: IGR J16318–4848 and GX 301–2

We present evidence for excess long-wavelength emission from two high-mass X-ray binaries, IGR J16318-4848 and GX 301-2, that show enormous obscuration (N_H ≃ 10^(23)-10^(24) cm^(-2)) in their X-ray spectra. Using archival near- and mid-infrared data, we show that the spectral energy distributions of IGR J16318-4848 and GX 301-2 are substantially higher in the mid-infrared than their expected stellar emission. We successfully fit the excesses with ~1000 K blackbodies, which suggests that they are due to warm circumstellar dust that also gives rise to the X-ray absorption. However, we need further observations to constrain the detailed properties of the excesses. This discovery highlights the importance of mid-infrared observations for understanding highly obscured X-ray binaries

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

Cognitively Engineering a Virtual Collaboration Environment for Crisis Response

Crisis response situations require collaboration across many different organizations with different backgrounds, training, procedures, and goals. The Indian Ocean Tsunami in 2004 and the Hurricane Katrina relief efforts in 2005 emphasized the importance of effective communication and collaboration. In the former, the Multinational Planning Augmentation Team (MPAT) supported brokering of requests for assistance with offers of help from rapidly deployed military and humanitarian assistance facilities. In the aftermath of Hurricane Katrina, the National Guard Soldiers and active component Army Soldiers assisted other state, federal, and non-government organizations with varying degrees of efficiency and expediency. Compounding the challenges associated with collaboration during crisis situations is the distributed nature of the supporting organizations and the lack of a designated leader across these military, government, nongovernment organizations. The Army Research Laboratory is collaborating with the University of Edinburgh, University o

Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localisation of polycystin-2 in vivo and in vitro

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca2 + -permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser812) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser76) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser76/Ser80) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology