Subcellular localization and signaling properties of dishevelled in developing vertebrate embryos

The Dishevelled protein mediates several diverse biological processes. Intriguingly, within the same tissues where Xenopus Dishevelled (Xdsh) controls cell fate via canonical Wnt signaling, it also controls cell polarity via the vertebrate planar cell polarity (PCP) cascade [1, 2, 3, 4, 5, 6, 7, 8 and 9]. The relationship between subcellular localization of Dishevelled and its signaling activities remains unclear; conflicting results have been reported depending upon the organism and cell types examined [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20]. We have approached this issue by developing new reagents to sequester wild-type Dishevelled protein either at the cell membrane or away from the cell membrane. Removal of Dishevelled from the cell membrane disrupts convergent extension by preventing Rho/Rac activation and mediolateral cell polarization. By manipulating the subcellular localization of K-->M (dsh1), we show that this mutation inhibits Dishevelled activation of Rac, regardless of its subcellular localization. These data demonstrate that membrane localization of Dishevelled is a prerequisite for vertebrate PCP signaling. However, both membrane-targeted and cytoplasm-targeted Dishevelled can potently activate canonical Wnt signaling, suggesting that local concentration of Dishevelled protein, but not its spatial localization, is central to canonical Wnt signaling. These results suggest that in vertebrate embryos, subcellular localization is insufficient to account for the pathway specificity of Dishevelled in the canonical Wnt versus PCP signaling cascades.     

Planar polarization of Drosophila and vertebrate epithelia

Our understanding of the actin and microtubule rearrangements that generate planar polarity in Drosophila and in vertebrate epithelia has been extended by recent discoveries. Three different Rho family proteins have been shown to mediate polarization in the wing and the eye of Drosophila. In vertebrates, the importance of myosin Vlla has been uncovered by mutations that cause defects in planar polarization in the ear. Advances in our understanding of the Frizzled pathway, which coordinates planar polarization in Drosophila, are moving the field closer to understanding the links between signal transduction and polarized cytoskeletal reorganization.     

Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.     

Purification, characterization, and localization of 70 kDa calcium-sensitive protein (calelectrin) from mammary glands

Mammary glands contain a group of calcium-sensitive proteins that bind to membranes in a calcium-dependent manner. Using the calcium-dependent binding to hydrophobic surfaces in combination with conventional techniques, we have purified the 70 kDa mammary calcium-binding protein (70 kDa M-CBP) to homogeneity. Antisera prepared to the 70 kDa M-CBP or to bovine liver 67 kDa calelectrin reacted in immunoblot analysis with the 70 kDa M-CBP antigen and with several additional mammary CBP species in crude tissue homogenates. Limited proteolysis of the 70 kDa M-CBP produced smaller immunoreactive species; extensive proteolysis resulted in more complete degradation of the protein. Identical data were obtained with digestion of 67 kDa calelectrin. The pl for the 70 kDa M-CBP was determined to be approximately 5.8; the same value reported for 67 kDa calelectrin. Phosphorylation of 70 kDa M-CBP was not detected in epithelial cell culture metabolic labeling. Immunohistochemical localization showed the protein to be located in ductal epithelia of virgin mouse mammary glands with a pattern of increased staining of the basal portions of the cells. Some stromal cells were also reactive. Apparently, the 70 kDa M-CBP and 67 kDa calelectrin are the same protein. Furthermore, like the 32.5 calelectrin (endonexin) and calpactin I/p36/lipocortin II, the 70 kDa protein appears to be a ductal epithelial cell associated protein in the mammary gland.     

Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.

Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.     

Streptococcal protein G. Gene structure and protein binding properties

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.     

Purification, characterization and antifungal properties of a lipid-transfer protein from sunflower (Helianthus annuus) seeds

An antifungal protein from Helianthus annuus L. seeds (Ha-AP10) has been purified to homogeneity and characterized. Ha-AP10 purification was performed by gel filtration, cation exchange chromatography and reverse phase HPLC. Its molecular mass was estimated to be 10 kDa and western blot analyses suggest that it has an extracellular location. The N-terminal sequence of Ha-AP10 showed strong homology to some plant lipid-transfer proteins (LTPs). Antifungal tests have demonstrated that Ha-AP10 exerts a fungistatic effect. It completely inhibits the germination of spores of the fungal pathogen Fusarium solani f. sp. eumartii at a concentration of 40 μg ml⁻¹ and produces a 50% growth inhibition at 6.5 μg ml⁻¹ (0.65 μ M ). These data place Ha-AP10 among the most potent antifungal LTPs described so far.     

Isolation and characterization of maize cDNAs encoding a high mobility group protein displaying a HMG-box.

cDNAs encoding a nonhistone chromosomal high mobility group (HMG) protein corresponding to the animal HMG1 family were isolated from a maize cDNA library using an immunoscreening approach. The cDNAs revealed an open reading frame of 471 base pairs together with 413 base pairs of flanking region, in agreement with the size of mRNA detected by Northern analysis of maize endosperm RNA. Like its animal counterparts the 17146 Da maize HMG protein contains a basic aminoterminus and an acidic carboxyterminus. The HMG-box region of this plant HMG protein shows striking sequence similarity to members of the vertebrate HMG1 family. Based on Southern blot hybridization analysis of genomic DNA, the isolated cDNA appears to be derived from a single or low copy gene.     

Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.     

Antibody to thymus myosin: its immunological characterization and use for immunocytochemical localization of myosin in vertebrate nonmuscle cells

Thymus myosin differs immunologically from smooth muscle and striated muscle myosin isoenzymes. In the enzyme linked immunosorbent assay a moderate degree of cross reaction was observed between anti-thymus myosin and myosin from chicken gizzard (about 50% of the titer of the homologous reaction). In contrast, the cross reactivity between thymus myosin and antibodies to gizzard myosin was very low (about 5%) and no significant cross reaction was observed between thymus myosin and antibodies to striated muscle myosin and vice versa (below 1%). Antibodies to thymus myosin were further distinguished from antibodies to gizzard and striated muscle myosin by their reaction with both smooth muscle and a very broad spectrum of vertebrate nonmuscle cells. Nonmuscle cells reacting with anti-thymus myosin included (1) cell types which did not display any detectable affinity for anti-gizzard myosin (e.g. lymphocytes, polymorphonuclear leucocytes, vascular endothelium, adrenal chromaffine cells) and (2) cell types which reacted with anti-gizzard myosin as well (e.g. intestinal epithelial brush border, thymic epithelial cells, liver cells and stress fibres of cultured cells). These results illustrate, that anti-thymus myosin is a potent tool for investigating the intracellular localization of myosin in most if not all vertebrate nonmuscle cells. With respect to lymphatic tissue the present findings indicate that lymphocyte maturation appears to be accompanied by an increased level of expression of myosin and filamentous actin (the latter was visualized by labelled phalloidin). On the ultrastructural level, gold labelled antibodies to thymus myosin bound preferentially to the head region of in vitro assembled thymus myosin filaments. In cultured cells (PtK1) the antibodies showed a particular affinity for stress fibre densities, and in lymphocytes the anti-myosin label (immunoperoxidase) displayed a more or less diffuse distribution which was similar to the distribution of actin filaments (identified by decoration with heavy meromyosin).     

Immunocytochemical localization of progesterone-binding protein (PBP) in guinea-pig placental tissue

Summary The cellular localization of progesterone-binding protein (PBP) in the guinea-pig placenta was studied by use of immunocytochemical procedures. Within the chorioallantoic placenta, a strong positive reaction was observed in the interlobar and marginal trophoblast from the third week of gestation to term. PBP was localized in the cytoplasm of the syncytiotrophoblast, and the nuclei were never stained. At the ultrastructural level, the immunoreaction was associated with the rough endoplasmic reticulum, the Golgi apparatus and the perinuclear space. No deposits were seen in any other cell organelles. This localization strongly suggests that the interlobar syncytium is related to PBP synthesis. In the labyrinth, a weak immunoreaction was observed by light microscopy around some blood lacunae. At the ultrastructural level the dense deposits were localized in vesicles located near the maternal lacunae.The distribution of PBP was also studied by light microscopy in other tissues from pregnant guinea-pig. No PBP, or PBP-like material, was detected inside cells from liver, muscle, heart, lung, kidney, ovary, and uterus. A weak immunoreaction for PBP was detected in vascularized zones of these organs.These observations strongly suggest that PBP, a protein related to gestation in the guinea-pig, is elaborated by the placental tissue of this hystricomorph rodent. PBP is the first steroid-binding plasma protein shown to be of extrahepatic origin.     

Structural characterization of the nickel-binding properties of Bacillus pasteurii urease accessory protein (Ure)E in solution

Urease activation is critical to the virulence of many human and animal pathogens. Urease possesses multiple, nickel-containing active sites, and UreE, the only nickel-binding protein among the urease accessory proteins, activates urease by transporting nickel ions. We performed NMR experiments to investigate the solution structure and the nickel-binding properties of Bacillus pasteurii (Bp) UreE. The secondary structures and global folds of BpUreE were determined for its metal-free and nickel-bound forms. The results indicated that no major structural change of BpUreE arises from the nickel binding. In addition to the previously identified nickel-binding site (Gly(97)-Cys(103)), the C-terminal tail region (Lys(141)-His(147)) was confirmed for the first time to be involved in the nickel binding. The C-terminally conserved sequence ((144)GHQH(147)) was confirmed to have an inherent nickel-binding ability. Nickel addition to 1.6 mm subunit, a concentration where BpUreE predominantly forms a tetramer upon the nickel binding, induced a biphasic spectral change consistent with binding of up to at least three nickel ions per tetrameric unit. In contrast, nickel addition to 0.1 mm subunit, a concentration at which the protein is primarily a dimer, caused a monophasic spectral change consistent with more than 1 equivalent per dimeric unit. Combined with the equilibrium dialysis results, which indicated 2.5 nickel equivalents binding per dimer at a micromolar protein concentration, the nickel-binding stoichiometry of BpUreE at a physiological concentration could be three nickel ions per dimer. Altogether, the present results provide the first detailed structural data concerning the nickel-binding properties of intact, wild-type BpUreE in solution.