Echinoid is an immunoglobulin domain-containing transmembrane protein that modulates cell-cell signaling by Notch and the EGF receptors. We show that, in the Drosophila wing disc epithelium, Echinoid is a component of adherens junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and beta-catenin (a DE-Cadherin interacting protein) each possess a C-terminal PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly position Bazooka to adherens junctions. Echinoid also links to actin filaments by binding to Canoe/AF-6/afadin. Moreover, interfaces between Echinoid- and Echinoid+ cells, like those between DE-Cadherin- and DE-Cadherin+ cells, are deficient in adherens junctions and form actin cables. These characteristics probably facilitate the strong sorting behavior of cells that lack either of these cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin accumulate a high density of the reciprocal protein, further suggesting that Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion.

Echinoid (Ed) is a homophilic immunoglobulin domain-containing cell adhesion molecule (CAM) that localizes to adherens junctions (AJs) and cooperates with Drosophila melanogaster epithelial (DE)-cadherin to mediate cell adhesion. Here we show that Ed takes part in many processes of dorsal closure, a morphogenetic movement driven by coordinated cell shape changes and migration of epidermal cells to cover the underlying amnioserosa. Ed is differentially expressed, appearing in epidermis but not in amnioserosa cells. Ed functions independently from the JNK signaling pathway and is required to regulate cell morphology, and for assembly of actomyosin cable, filopodial protrusion and coordinated cell migration in dorsal-most epidermal cells. The effect of Ed on cell morphology requires the presence of the intracellular domain (Ed(intra)). Interestingly, Ed forms homodimers in vivo and Ed(intra) monomer directly associates with unconventional myosin VI/Jaguar (Jar) motor protein. We further show that ed genetically interacts with jar to control cell morphology. It has previously been shown that myosin VI is monomeric in vitro and that its dimeric form can associate with and travel processively along actin filaments. Thus, we propose that Ed mediates the dimerization of myosin VI/Jar in vivo which in turn regulates the reorganization and/or contraction of actin filaments to control changes in cell shape. Consistent with this, we found that ectopic ed expression in the amnioserosa induces myosin VI/Jar-dependent apical constriction of this tissue.

Background: Myb1 mediates transcription suppression of an ap65-1 gene. Results: TvCyP1 may accelerate conformational changes in Myb1 to enable its release from certain vesicles. Conclusion: TvCyP1 is essential for moving Myb1 toward the nucleus. Significance: This study elucidates a key step in nuclear translocation of Myb1 and provides tools to study the physiological role of TvCyP1.

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH 2 , designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an ␣-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH 2 , designated Pac-525 rev , also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.

No abstract

Antimicrobial peptides are important components of the host innate defense mechanism against invading pathogens, especially for drug-resistant bacteria. In addition to bactericidal activity, the 25 residue peptide TP4 isolated from Nile tilapia also stimulates cell proliferation and regulates the innate immune system in mice. In this report, TP4 hyperpolarized and depolarized the membrane potential of Pseudomonas aeruginosa at sub-lethal and lethal concentrations. It also inhibited and eradicated biofilm formation. The in vitro binding of TP4 to bacterial outer membrane target protein, OprI, was markedly enhanced by a membranelike surfactant sarkosyl and lipopolysaccharide, which converted TP4 into an α-helix. The solution structure of TP4 in dodecylphosphocholine was solved by NMR analyses. It contained a typical α-helix at residues Phe10-Arg22 and a distorted helical segment at Ile6-Phe10, as well as a hydrophobic core at the N-terminus and a cationic patch at the C-terminus. Residues Ile16, Leu19 and Ile20 in the hydrophobic face of the main helix were critical for the integrity of amphipathic structure, other hydrophobic residues played important roles in hemolytic and bactericidal activities. A model for the assembly of helical TP4 embedded in sarkosyl vesicle is proposed. This study may provide valuable insight for engineering AMPs to have potent bactericidal activity but low hemolytic activity.

cIn Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNAbinding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

SUMMARYCell sorting involves the segregation of two cell populations into 'immiscible' adjacent tissues with smooth borders. Echinoid (Ed), a nectin ortholog, is an adherens junction protein in Drosophila, and cells mutant for ed sort out from the surrounding wild-type cells. However, it remains unknown which factors trigger cell sorting. Here, we dissect the sequence of this process and find that cell sorting occurs when differential expression of Ed triggers the assembly of actomyosin cable. Conversely, Ed-mediated cell sorting can be rescued by recruitment of Ed, via homophilic or heterophilic interactions, to the wild-type cell side of the clonal interface, even when differential Ed expression persists. We found, unexpectedly, that when actomyosin cable was largely absent, differential adhesion was sufficient to cause limited cell segregation but with a jagged tissue border (imperfect sorting). We propose that Ed-mediated cell sorting is driven both by differential Ed adhesion that induces cell segregation with a jagged border and by actomyosin cable assembly at the interface that smoothens this border.