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Results for “spatial domain identification”

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PubMed1983

Identification of epitopes on the E glycoprotein of Saint Louis encephalitis virus using monoclonal antibodies.

Twenty-one hybridomas producing monoclonal antibodies specific for the E glycoprotein of St. Louis encephalitis (SLE) virus, strain MSI-7, have been isolated. Serologic reactivities were initially determined by cross-reactivity indirect immunofluorescence assays using 22 strains of SLE virus and 8 other related flaviviruses. Four groups demonstrating type-, subcomplex-, supercomplex-, and group-specific reactivity patterns were identified. Analysis of hemagglutination-inhibition (HI) and virus neutralization (N) subdivided the cross-reactivity groups into eight epitopes (E-1a,b,c,d, E-2, E-3, and E-4a,b). The antibodies could detect strain differences between SLE viruses isolated from various geographic areas. Analysis of the spatial arrangements of these epitopes using competitive binding assays with representative antibodies possessing similar binding avidities, indicated that the protein was a continuum of six overlapping domains.

Antibodies, Monoclonal↗

PubMed1989

Molecular model of the G protein alpha subunit based on the crystal structure of the HRAS protein.

A structural model of guanine nucleotide-binding regulatory protein alpha subunits (G alpha subunits) is proposed based on the crystal structure of the catalytic domain of the human HRAS protein (p21ras). Because of low overall sequence similarity, structural and functional constraints were used to align the G alpha consensus sequence with that of p21ras. The resulting G alpha model specifies the spatial relationship among the guanine nucleotide-binding site, the binding site of the beta gamma subunit complex, likely regions of effector and receptor interaction, and sites of cholera and pertussis toxin modification. The locations in the model of the experimentally determined sites of proteolytic digestion, point mutation, monoclonal antibody binding, and toxin modification are consistent with and help explain the observed biological activity. Two important findings from our model are (i) the orientation of the G alpha model with respect to the membrane and (ii) the identification of the spatial proximity of the N- and C-terminal regions. Furthermore, by analogy to p21ras, the model assigns specific residues in G alpha required for binding the guanosine (G-box) and phosphates (PO4-box) and identifies residues potentially involved in the conformational switch mechanism (S-box). Specification of these critical regions in the G alpha model suggests guidelines for construction of mutants and chimeric proteins to experimentally test structural and functional hypotheses.

Adenosine Diphosphate Ribose↗

PubMed1988

Calculation of the three-dimensional structure of Saccharomyces cerevisiae cytochrome b inserted in a lipid matrix.

Cytochrome b is an integral membrane protein, which forms the core of the ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex. A computer-aided three-dimensional modeling procedure was carried out in four steps. First, the candidate hydrophobic helices were searched for throughout the protein primary sequence by a computer procedure based upon the method of Eisenberg; second, a secondary helical structure was imposed to the transmembrane peptides; third, the helical segments at a lipid-water interface were oriented, and finally the possible interactions between helices with similar properties were investigated. This procedure enabled the identification of nine hydrophobic segments, of which eight are membrane-spanning helices while one has amphipathic properties. Three hydrophilic receptor-binding domains were also identified. Based upon their hydrophobicity profiles, the transmembrane helices could be associated in pairs inside the lipid bilayer. In our folding model proposed for cytochrome b, all mutation sites are not only located on the same side of the membrane but are also in close proximity in the three-dimensional structure. Inhibitor resistance mutational sites which were recently characterized (di Rago, J.-P., and Colson, A.-M. (1988) J. Biol. Chem. 263, 12564-12570) have been located on this model. Moreover, the receptor-binding domains and the mutation sites are close neighbors in the three-dimensional spatial representation.

Amino Acid Sequence↗

PubMed1994

Identification and cloning of ELF-1, a developmentally expressed ligand for the Mek4 and Sek receptor tyrosine kinases.

Mek4 and Sek are tyrosine kinases with expression patterns in the mouse embryo that suggest important functions in early development. However, like all Eph family kinases, both were identified as orphan receptors without known ligands. We show that Mek4 and Sek soluble receptor-alkaline phosphatase fusion proteins can be used in a procedure termed RAP in situ to identify regions of ligand expression in the mouse embryo. Based on this spatial information, a cDNA expression library was prepared, and was screened with the fusion proteins to identify Eph ligand family-1 (ELF-1). In cell lines and embryos, ELF-1 is membrane bound by a phosphatidylinositol tail, a feature that may account for unique biological functions. Its sequence is homologous with B61, a ligand for the Eck kinase, defining a family of related ligands. The expression domains of ELF-1, Mek4 and Sek indicate potential roles in embryonic patterning.

Amino Acid Sequence↗

PubMed2002

Changes in connexin43 in early ocular surface development.

PURPOSE: In the limbo-corneal epithelium the stem and early precursor epithelial cell pool is confined to the limbal rim. Among the features associated with this spatial segregation is the general paucity of connexin43 (Cx43) within the limbal basal cell population and its complete absence in resident stem cells. The limbo-corneal epithelial lineage derives from a Cx43-positive (Cx43+) embryonic outer ectoderm. Accordingly, as a means of identifying the process through which limbal cell phenotypes emerge, we investigated the expression of Cx43 in the ocular surface of embryonic rats. METHODS: Ocular surface expression of Cx43 or K12 was determined in cryostat sections of rat embryos and eyes using immunohistological methods. RESULTS: Changes in Cx43 expression revealed the early phenotypic divergence of three main epithelial cell phenotypes of the ocular surface. An analysis of the level and distribution pattern of Cx43 puncta lead to the identification of two distinct domains by embryonic day 10 (E10), a stage that occurs soon after formation of the lens vesicle. Additionally, at E12, ectodermal cells directly adjacent to the edges of the developing retina no longer express connexin. A comparison of anatomical and expression changes throughout embryonic development demonstrated that the two early zones represent the rudiments for the epithelia of the central cornea and conjunctiva, respectively, and that the isolated Cx43-negative (Cx43-) cells represent the precursors of the basal and, putatively, stem cells of the limbal epithelium. CONCLUSIONS: Changes in Cx43 expression revealed that the phenotypic divergence of ocular surface epithelial cells and the generation of limbo-corneal stem cell precursors takes place at a very early stage in ocular development, ahead of the establishment of any identifiable anatomical or differentiation features for these domains.

Animals↗

PubMed1988

Communication compartments in the gastrulating mouse embryo.

We characterized the pattern of gap junctional communication in the 7.5-d mouse embryo (at the primitive streak or gastrulation stage). First we examined the pattern of dye coupling by injecting the fluorescent tracers, Lucifer Yellow or carboxyfluorescein, and monitoring the extent of dye spread. These studies revealed that cells within all three germ layers are well coupled, as the injected dye usually spread rapidly from the site of impalement into the neighboring cells. The dye spread, however, appeared to be restricted at specific regions of the embryo. Further thick section histological analysis revealed little or no dye transfer between germ layers, indicating that each is a separate communication compartment. The pattern of dye movement within the embryonic ectoderm and mesoderm further suggested that cells in each of these germ layers may be subdivided into smaller communication compartments, the most striking of which are a number of "box-like" domains. Such compartments, unlike the restrictions observed between germ layers, are consistently only partially restrictive. In light of these results, we further monitored ionic coupling to determine if some coupling might nevertheless persist between germ layers. For these studies, Lucifer Yellow was coinjected while ionic coupling was monitored. The injected Lucifer Yellow facilitated the identification of the impalement sites, both in the live specimen and in thick sections in the subsequent histological analysis. By using this approach, all three germ layers were shown to be ionically coupled, indicating that gap junctional communication is maintained across the otherwise dye-uncoupled "germ layer compartments." Thus our results demonstrate that partially restrictive communication compartments are associated with the delamination of germ layers in the gastrulating mouse embryo. The spatial distribution of these compartments are consistent with a possible role in the underlying development.

Animals↗

PubMed1995

Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5' regulatory region.

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5' end cDNA and 5' regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (-1007 to -828 and -455 to -101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3' region, extending from the transcriptional start site to part. of intron 2, must be present in all the DNA constructs used.

Aging↗

PubMed1997

Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection.

We describe here the cloning of mouse complementary DNAs encoding a novel protein, Rb-8 neural cell adhesion molecule (RNCAM), with a predicted extracellular region of five immunoglobulin C2-type domains followed by two fibronectin type III domains. Alternative splicing is likely to generate two RNCAM isoforms, which are differently attached to the cell membrane. These structural features and overall sequence identity identify this protein as a novel member of a cell adhesion molecule subgroup together with vertebrate neural cell adhesion molecule, Aplysia cell adhesion molecule, and Drosophila fasciclin II. In insects, fasciclin II is present on a restricted subset of embryonic central nervous system axons where it controls selective axon fasciculation. Intriguingly, RNCAM likewise is expressed in subsets of olfactory and vomeronasal neurons with topographically defined axonal projections. The spatial expression RNCAM corresponds precisely to that of certain odorant receptor expression zones of the olfactory epithelium. These expression patterns thus render RNCAM the first described cell adhesion molecule with a potential regulatory role in formation of selective axonal projections important for olfactory sensory information coding.

Alternative Splicing↗

PubMed2003

The T-box-encoding Dorsocross genes function in amnioserosa development and the patterning of the dorsolateral germ band downstream of Dpp.

Dpp signals are responsible for establishing a variety of cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although we have a reasonably clear picture of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. In the present study, we describe three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, which are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. We show that these genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, we demonstrate that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, we show that the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend our knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals.

Amino Acid Sequence↗

PubMed1999

Identification of a Drosophila homologue to vertebrate Crk by interaction with MBC.

The vertebrate adapter protein termed Crk was initially identified from the chicken CT10 retrovirus on the basis of its transforming activity (Mayer et al., 1988. Nature 332, 272-275). We have identified a Drosophila protein with homology to vertebrate Crk, termed dCRK, by interaction with the protein encoded by the Drosophila myoblast city (mbc) gene. The dCRK protein has extensive homology to the both the Crk-II form of vertebrate Crk and the Crk-related protein CRKL, and includes one SH2 domain followed by two SH3 domains. A single protein of approx. 37kDa is detected in extracts from embryos, and Northern analysis revealed a single transcript of 1.3kb. The dCrk mRNA is abundant throughout embryogenesis, declines during the larval stages, and reappears during pupation. In addition, maternally-provided transcripts have been detected. During embryogenesis, the spatial distribution of this transcript is relatively broad and appears to include all germ layers. Finally, dCrk is located on the fourth chromosome, approximately at cytological position 101F-102A.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Detecting microdomains in intact cell membranes.

Current models for cellular plasma membranes focus on spatial heterogeneity and how this heterogeneity relates to cell function. In particular, putative lipid raft membrane domains have been postulated to exist based in large part on the results that a significant fraction of the membrane is detergent insoluble and that molecules facilitating key membrane processes like signal transduction are often found in the detergent-resistant membrane fraction. Yet, the in vivo existence of lipid rafts remains extremely controversial because, despite being sought for more than a decade, evidence for their presence in intact cell membranes is inconclusive. In this review, a variety of experimental techniques that have been or might be used to look for lipid microdomains in intact cell membranes are described. Experimental results are highlighted and the strengths and limitations of different techniques for microdomain identification and characterization are assessed.

Biomarkers↗

PubMed2002

Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes.

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.

Amino Acid Sequence↗

PubMed1999

Subcellular localization of full-length and truncated Trk receptor isoforms in polarized neurons and epithelial cells.

Neurotrophins affect neuronal development and plasticity via spatially localized effects, yet little is known about the subcellular distribution of the Trk neurotrophin receptors and the impact of this distribution on neurotrophin action. To address this, we examined the subcellular location of full-length TrkB and TrkC tyrosine kinase receptors and truncated TrkB isoforms after transfection of Madin-Darby canine kidney (MDCK) cells, dissociated primary hippocampal neurons, and cortical neurons within intact brain slices. Myc-, herpes virus glycoprotein (HVG)-, or FLAG-derived epitope-tagged receptor isoforms were created to allow their unambiguous identification and localization after transfection. All tagged receptors were appropriately synthesized, and full-length myc-TrkB and myc-TrkC mediated appropriate neurotrophin-signaling events. We found that full-length TrkB receptors were excluded from the apical domain of MDCK cells but that TrkC receptors were present in both apical and basolateral domains. Full-length TrkB and TrkC were found throughout transfected primary cultured hippocampal neurons and transfected neurons in neocortical brain slices and showed no evidence of vectorial sorting. Truncated forms of TrkB were also homogeneously distributed in MDCK cells, dissociated hippocampal neurons, and cortical neurons within slice preparations. Levels of full-length and truncated TrkB were examined in postsynaptic densities; both receptor isoforms were present but only moderately enriched in these structures. Together, these findings suggest that Trk receptors are uniformly distributed in both axonal and dendritic compartments and that local neurotrophin responses are controlled by other mechanisms.

Amino Acid Sequence↗

PubMed2004

HthA, a putative DNA-binding protein, and HthB are important for fruiting body morphogenesis in Myxococcus xanthus.

In response to starvation, Myxococcus xanthus initiates a developmental programme that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation and involves temporally and spatially coordinated changes in gene expression. This paper reports the identification of two genes, hthA and hthB, that are important for fruiting body formation. hthA and hthB are co-transcribed, and transcription of the two genes decreases strongly during development. Loss of HthA and HthB function results in delayed aggregation, a reduction in the level of sporulation, and abnormal developmental gene expression. Extracellular complementation experiments showed that the developmental defects caused by loss of HthA and HthB function are not due to the inability to synthesize an intercellular signal required for fruiting body formation. HthA, independent of HthB, is required for aggregation. HthB, alone or in combination with HthA, is required for sporulation. HthA is predicted to contain a C-terminal helix-turn-helix DNA-binding domain. Intriguingly, the N-terminal part of HthA does not exhibit significant amino acid similarity to proteins in the databases. The HthB protein lacks homologues in the databases. The results suggest that HthA is a novel DNA-binding protein, which regulates transcription of genes important for aggregation, and that HthB, alone or in combination with HthA, stimulates sporulation.

Amino Acid Sequence↗

PubMed2004

The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins.

The vulval precursor cells (VPCs) are spatially patterned by a LET-23/EGF receptor-mediated inductive signal and a LIN-12/Notch-mediated lateral signal. The lateral signal has eluded identification, so the mechanism by which lateral signaling is activated has not been known. Here, we computationally identify ten genes that encode potential ligands for LIN-12, and show that three of these genes, apx-1, dsl-1, and lag-2, are functionally redundant components of the lateral signal. We also show that transcription of all three genes is initiated or upregulated in VPCs in response to inductive signaling, suggesting that direct transcriptional control of the lateral signal by the inductive signal is part of the mechanism by which these cell signaling events are coordinated. In addition, we show that DSL-1, which lacks a predicted transmembrane domain, is a natural secreted ligand and can substitute for the transmembrane ligand LAG-2 in different functional assays.

Animals↗

PubMed2003

Actions blind to conceptually overlapping stimuli.

Participants are worse at identifying spatial symbols (arrowheads) while performing spatially compatible manual key presses. The present experiments investigated the generality of this "blindness effect" to response-compatible stimuli. In Experiment 1 a left key press deteriorated the identification of left-pointing arrows, and a right key press deteriorated the perception of right-pointing arrows, independent of the hands used to press the key. Thus the blindness effect is based on codes of the distal response location rather than on the body-intrinsic anatomical connection of the hands. Experiment 2 extended the blindness effect to verbal responses and written position words (left, right, up, down). Vocalizing a position word blinded to directly compatible position words (e.g., left-left), but not to orthogonally compatible position words (e.g., left-down). This result suggests that the use of identical stimulus-response codes, and not the use of saliency-matching but distinct codes, suffices to produce blindness effects. Finally, Experiment 3 extended the blindness phenomenon beyond the spatial domain by demonstrating blindness between saying color words and perceiving color patches. Altogether, the experiments revealed action-induced blindness to be a phenomenon of broad empirical validity occurring whenever action and perception afford simultaneous access to the same conceptual codes.

Adolescent↗

PubMed2002

M13 endopeptidases: New conserved motifs correlated with structure, and simultaneous phylogenetic occurrence of PHEX and the bony fish.

M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures. Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis. This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome). New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated. Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures. Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue. Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX. Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates.

Amino Acid Motifs↗

PubMed2002

A transgenic Lef1/beta-catenin-dependent reporter is expressed in spatially restricted domains throughout zebrafish development.

The Wnt/beta-catenin signaling pathway plays multiple roles during embryonic development, only a few of which have been extensively characterized. Although domains of Wnt expression have been identified throughout embryogenesis, anatomical and molecular characterization of responding cells has been mostly unexplored. We have generated a transgenic zebrafish line that expresses a destabilized green fluorescent protein (GFP) variant under the control of a beta-catenin responsive promoter. Early zygotic expression of this transgene (TOPdGFP) mirrors known domains of Wnt signaling in the embryo. Loss of Lef1 activity results in decreased reporter expression and posterior defects, while loss of Tcf3 (Headless, Hdl) activity does not alter reporter expression, even though it results in loss of forebrain structures. In addition, ectopic Wnt1 expression can activate the reporter. In older embryos, we identify a number of transgene-expressing cell populations as novel sites of beta-catenin signaling. We conclude that our TOP-dGFP reporter line faithfully illustrates domains of beta-catenin activity and enables the identification of responsive cell populations.

Animals↗