Binding to F-actin guides cadherin cluster assembly,stability, and movement

The cadherin extracellular region produces intercellular adhesion clusters through trans- and cis-intercadherin bonds, and the intracellular region connects these clusters to the cytoskeleton. To elucidate the interdependence of these binding events, cadherin adhesion was reconstructed from the minimal number of structural elements. F-actin–uncoupled adhesive clusters displayed high instability and random motion. Their assembly required a cadherin cis-binding interface. Coupling these clusters with F-actin through an α-catenin actin-binding domain (αABD) dramatically extended cluster lifetime and conferred direction to cluster motility. In addition, αABD partially lifted the requirement for the cis-interface for cluster assembly. Even more dramatic enhancement of cadherin clustering was observed if αABD was joined with cadherin through a flexible linker or if it was replaced with an actin-binding domain of utrophin. These data present direct evidence that binding to F-actin stabilizes cadherin clusters and cooperates with the cis-interface in cadherin clustering. Such cooperation apparently synchronizes extracellular and intracellular binding events in the process of adherens junction assembly.     

G-protein-coupled signals control cortical actin assembly by controlling cadherin expression in the early Xenopus embryo

During embryonic development, each cell of a multicellular organ rudiment polymerizes its cytoskeletal elements in an amount and pattern that gives the whole cellular population its characteristic shape and mechanical properties. How does each cell know how to do this? We have used the Xenopus blastula as a model system to study this problem. Previous work has shown that the cortical actin network is required to maintain shape and rigidity of the whole embryo, and its assembly is coordinated throughout the embryo by signaling through G-protein-coupled receptors. In this paper, we show that the cortical actin network colocalizes with foci of cadherin expressed on the cell surface. We then show that cell-surface cadherin expression is both necessary and sufficient for cortical actin assembly and requires the associated catenin p120 for this function. Finally, we show that the previously identified G-protein-coupled receptors control cortical actin assembly by controlling the amount of cadherin expressed on the cell surface. This identifies a novel mechanism for control of cortical actin assembly during development that might be shared by many multicellular arrays.     

ATP-dependent membrane assembly of F-actin facilitates membrane fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.     

An atlas of differential gene expression during early Xenopus embryogenesis

We have carried out a large-scale, semi-automated whole-mount in situ hybridization screen of 8369 cDNA clones in Xenopus laevis embryos. We confirm that differential gene expression is prevalent during embryogenesis since 24% of the clones are expressed non-ubiquitously and 8% are organ or cell type specific marker genes. Sequence analysis and clustering yielded 723 unique genes displaying a differential expression pattern. Of these, 18% were already described in Xenopus, 47% have homologs and 35% are lacking significant sequence similarity in databases. Many of them encode known developmental regulators. We classified 363 of the 723 genes for which a Gene Ontology annotation for molecular function could be attributed and found 'DNA binding' and 'enzyme' the most represented terms. The most common protein domains encoded in these embryonic, differentially expressed genes are the homeobox and RNA Recognition Motif (RRM). Fifty-nine putative orthologs of human disease genes, and 254 organ or cell specific marker genes were identified. Markers were found for nasal placode and archenteron roof, organs for which a specific marker was previously unavailable. Markers were also found for novel subdomains of various other organs. The tissues for which most markers were found are muscle and epidermis. Expression of cell cycle regulators fell in two classes, containing proliferation-promoting and anti-proliferative genes, respectively. We identified 66 new members of the BMP4, chromatin, endoplasmic reticulum, and karyopherin synexpression groups, thus providing a first glimpse of their probable cellular roles. Cluster analysis of tissues to measure tissue relatedness yielded some unorthodox affinities besides expectable lineage relationships. In conclusion, this study represents an atlas of gene expression patterns, which reveals embryonic regionalization, provides novel marker genes, and makes predictions about the functional role of unknown genes.     

Regulation of Op18 during spindle assembly in Xenopus egg extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.     

Regulation and role of PDGF receptor alpha-subunit expression during embryogenesis.

The platelet-derived growth factor receptor alpha-subunit (PDGFR alpha) is the form of the PDGF receptor that is required for binding of PDGF A-chain. Expression of PDGFR alpha within the early embryo is first detected as the mesoderm forms, and remains characteristic of many mesodermal derivatives during later development. By 9.5 days of development, embryos homozygous for the Patch mutation (a deletion of the PDGFR alpha) display obvious growth retardation and deficiencies in mesodermal structures, resulting in the death of more than half of these embryos. Mutant embryos that survive this first critical period are viable until a new set of defects become apparent in most connective tissues. For example, the skin is missing the dermis and connective tissue components are reduced in many organs. By this stage, expression of PDGFR alpha mRNA is also found in neural crest-derived mesenchyme, and late embryonic defects are associated with both mesodermal and neural crest derivatives. Except for the neural crest, the lens and choroid plexus, PDGFR alpha mRNA is not detected in ectodermal derivatives until late in development in the central nervous system. Expression is not detected in any embryonic endodermal derivative at any stage of development. These results demonstrate that PDGFR alpha is differentially expressed during development and that this expression is necessary for the development of specific tissues.     

Regulation of neuronal SNARE assembly by the membrane

In the neuron, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) assembly acts centrally in driving membrane fusion, a required process for neurotransmitter release. In the cytoplasm, vesicular SNARE VAMP-2 (vesicle-associated membrane protein-2) engages with two plasma membrane SNAREs, syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa), to form the core complex that bridges two membranes. Although various factors regulate SNARE assembly, the membrane also aids in regulation by trapping VAMP-2 in the membrane. Fluorescence and EPR analyses revealed that the insertion of seven C-terminal core-forming residues into the membrane controls complex formation of the entire core region, even though the preceding 54 core-forming residues are fully exposed and freely moving. When two interfacial tryptophan residues in this region were replaced with hydrophilic serine residues, the mutation supported rapid complex formation. The results suggest that the membrane-proximal region of VAMP-2 is a regulatory module for SNARE assembly, providing new insights into calcium-triggered membrane fusion.     

Regulation of cadherin expression in nervous system development

This review addresses our current understanding of the regulatory mechanisms for classical cadherin expression during development of the vertebrate nervous system. The complexity of the spatial and temporal expression patterns is linked to morphogenic and functional roles in the developing nervous system. While the regulatory networks controlling cadherin expression are not well understood, it is likely that the multiple signaling pathways active in the development of particular domains also regulate the specific cadherins expressed at that time and location. With the growing understanding of the broader roles of cadherins in cell–cell adhesion and non-adhesion processes, it is important to understand both the upstream regulation of cadherin expression and the downstream effects of specific cadherins within their cellular context.     

Expression of Xenopus Daz-like protein during gametogenesis and embryogenesis

Using a monoclonal antibody raised against Xenopus Daz-like protein (Xdazl), we showed that Xdazl is present in all stages of male and female germ cells except mature spermatozoa. Xdazl is not localized to any specific regions in early-stage embryos, in contrast to the strict localization of its mRNA in the germ plasm. Xdazl disappears after gastrulation but reappears in the primordial germ cells situated at the genital ridge. This is the first detailed report on the protein expression of a Daz-like gene during gametogenesis and embryogenesis in Xenopus, showing the difference in expression patterns of its mRNA and protein.     

Expression of XBcad, a novel cadherin, during oogenesis and early development of Xenopus.

A gastrula cDNA library was screened using a cDNA probe encoding the cytoplasmic domain of uvomorulin, a mouse Ca(2+)-dependent cell adhesion molecule. A Xenopus cDNA clone was isolated, which shares an amino acid sequence identity with uvomorulin of 91% in the transmembrane and 89% in the cytoplasmic domain. A restriction fragment of 397 bp representing the lowest degree of identity to all other known cadherin sequences was used to study the expression pattern of this Xenopus cadherin gene on RNA and protein level. The 397 bp restriction fragment was expressed bacterially as fusion protein, against which polyclonal antibodies were raised. An mRNA of 3.9 kb and a corresponding 125 kDa glycoprotein could be identified. Both molecules are present throughout oogenesis and early embryogenesis. When cleavage starts, the protein becomes integrated into the newly formed membranes. This polypeptide is found at cell membranes of all blastomeres except those at the outer surface of the embryo. Immunoblots and immunohistological analyses of adult organs reveal that this protein is expressed in pituitary gland, lung and kidney. It could not be detected in liver, heart and skeletal muscle. Since this cadherin differs in its tissue distribution from that of U-cadherin and in sequence alignments from ep-cadherin, it was termed XBcad for Xenopus blastomere cadherin.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.