During Drosophila embryogenesis, the ventral epidermis dorsally expands and the left and right epithelial sheets meet and fuse along the dorsal midline. For this dorsal closure to occur, two PDZ domain proteins, Cno and ZO-1, are required. The dorsal epidermis remains open when the expression of ZO-1 and Cno are reduced simultaneously by hypomorphic mutations in the relevant loci. ZO-1 and Cno colocalize at adherens junctions in embryonic epithelia, and form a protein complex upon binding to each other. Genetic analysis showed that Cno is involved in the Jun N-terminal kinase (JNK) pathway for dorsal closure, as a modulator acting upstream of, or in parallel with, the small GTPase Drac1. The ZO-1-Cno complex may be involved in dynamic changes in cytoskeletal organization and cell adhesion during morphogenetic events associated with dorsal closure in the Drosophila embryo.

Cortactin is an actin filament-binding protein localizing at cortical regions of cells and a prominent substrate for Src family protein-tyrosine kinases in response to multiple extracellular stimuli. Human cortactin has been identified as a protein product of a putative oncogene, EMS1. In this report, we describe the identification of a Drosophila homolog of cortactin as a molecule that interacts with Drosophila ZO-1 using yeast twohybrid screening. Drosophila cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. Deletion and substitution mutant analyses revealed that the SH3 domain of Drosophila cortactin binds to a PXXP motif in the proline-rich domain of Drosophila ZO-1. Colocalization of these proteins at cell-cell junction sites was evident under a confocal laser-scanning microscope. In vivo association was confirmed by coimmunoprecipitation of cortactin and ZO-1 from Drosophila embryo lysates. We also demonstrate an association for each of the murine homologs by immunoprecipitation analyses of mouse tissue lysates. Our previous work has demonstrated the involvement of ZO-1 in a signaling pathway that regulates expression of the emc gene in Drosophila. The potential roles of the cortactin⅐ZO-1 complex in cell adhesion and cell signaling are discussed.Cell-cell adhesions are essential for the development of the multicellular organisms. Among the proteins composing the cell-cell adhesion complexes, members of the membrane-associated guanylate kinase homologs (MAGUKs) 1 are widely found in Hydra, Caenorhabditis elegans, Drosophila, and mammals (1-3). MAGUKs have distinctive domains including one or three copies of the PDZ domain, an SH3 domain, and a domain homologous to guanylate kinase (GUK) and implicated in both formation of cell-cell junctions and signal transduction. One of the most intensively characterized members of the MAGUKs is the mammalian ZO-1, which is known to associate with several cellular proteins including the components of cell-cell junctions (occludin, ␤-catenin, and ZO-2) and the components of cytoskeletal networks (␣-spectrin and actin filaments (F-actin)) (4 -9). While ZO-1 has been considered as a homolog of a Drosophila tumor suppresser Dlg, its biological functions in the cell-cell junction and signal transduction remain obscure (10, 11).We recently identified a new Drosophila MAGUK protein, Tamou, and reported its significant homology with ZO-1 (12). We will refer to Tamou as Drosophila ZO-1 (DZO-1) because we also found that the transgenes of mouse ZO-1 could replace the tam gene function in Drosophila.2 The DZO-1 tam1 mutant flies exhibit the supernumerary mechanosensory organs. This is a similar phenotype to that of an extramacrochaetae (emc) mutation. The emc gene encodes a helix-loop-helix type transcriptional regulator and negatively regulates specification of sensory organ precursor cells (13-16). We have previously shown that DZO-1 locates at cell-cell junctions and is involved in the signaling p...

Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.

Deregulated V(D)J recombination-mediated chromosomal rearrangements are implicated in the etiology of B-and T-cell lymphomagenesis. We describe three pathways for the formation of 5-deletions of the Notch1 gene in thymic lymphomas of wild-type or V(D)J recombination-defective severe combined immune deficiency (scid) mice. A pair of recombination signal sequence-like sequences composed of heptamer-and nonamer-like motifs separated by 12-or 23-bp spacers (12-and 23-recombination signal sequence) were present in the vicinity of the deletion breakpoints in wild-type thymic lymphomas, accompanied by palindromic or nontemplated nucleotides at the junctions. In scid thymic lymphomas, the deletions at the recombination signal sequence-like sequences occurred at a significantly lower frequency than in wild-type mice, whereas the deletions did not occur in Rag2 ؊/؊ thymocytes. These results show that the 5-deletions are formed by Rag-mediated V(D)J recombination machinery at cryptic recombination signal sequences in the Notch1 locus. In contrast, one third of the deletions in radiation-induced scid thymic lymphomas had microhomology at both ends, indicating that in the absence of DNAdependent protein kinase-dependent nonhomologous end-joining, the microhomology-mediated nonhomologous end-joining pathway functions as the main mechanism to produce deletions. Furthermore, the deletions were induced via a coupled pathway between Rag-mediated cleavage at a cryptic recombination signal sequence and microhomology-mediated endjoining in radiation-induced scid thymic lymphomas. As the deletions at cryptic recombination signal sequences occur spontaneously, microhomology-mediated pathways might participate mainly in radiation-induced lymphomagenesis. Recombination signal sequence-mediated deletions were present clonally in the thymocyte population, suggesting that thymocytes with a 5-deletion of the Notch1 gene have a growth advantage and are involved in lymphomagenesis.

The nuclear foci of phosphorylated histone H2AX (γH2AX) are frequently used as a marker for DNA double-strand breaks (DSBs) following ionizing radiation (IR). However, recent studies reported that γH2AX foci do not necessarily correlate with DSBs under other conditions. We showed that γH2AX foci induced by oxidative stress in hydrogen peroxide (H2O2)-treated cells displayed several different features from those induced by IR. The magnitude of γH2AX induction was heterogeneous among H2O2-treated cells. Some cells expressed small discrete γH2AX foci, whereas others expressed a gross γH2AX signal that was distributed throughout the nucleus. Oxidative stress-induced γH2AX was eliminated in DSB repair-deficient mutant cells as efficiently as in wild-type cells and was not necessarily accompanied by phosphorylated ataxia telangiectasia mutated (ATM) or 53BP1 foci. Analyses using specific inhibitors showed that ATM- and Rad3-related (ATR), rather than ATM, was the prominent kinase mediating the oxidative stress response. These results suggest that a major fraction of γH2AX induced by oxidative stress is not associated with DSBs. Single-stranded DNA arisen from stalled replication forks can cause the ATR-mediated induction of γH2AX. However, oxidative stress appeared to induce γH2AX in both S- and non-S-phase cells. These results suggest that there may be another pathway leading to the ATR-mediated induction of γH2AX in non-S-phase cells without DSBs.

Notch1 protein is a transmembrane receptor that directs various cell fate decisions. Active forms of Notch1 consisting of a transmembrane domain and an intracellular domain (Notch1TM) or only an intracellular domain (Notch1IC) function as oncoproteins. To elucidate the effect of Notch1 abnormalities in radiation-induced lymphomagenesis, we determined the structure of the Notch1 gene and examined the frequency and the sites of Notch1 rearrangements in radiation-induced mouse thymic lymphomas. The Notch1 gene consists of 37 exons, including three exons upstream of the previously reported exon 1. The transcript starting from exon 1 was the major transcript whereas the transcripts read upstream from exon 1a, in which amino acid sequences in the N-terminal region were changed, were minor. More than 50% of radiation-induced thymic lymphomas exhibited Notch1 rearrangements, suggesting that Notch1 acts as a major oncogene in radiation-induced lymphomagenesis. We identified three rearranged sites: novel sites in the 5' end region encompassing exons 1 and 2, the previously identified juxtamembrane extracellular region, and the 3' end region. The 5' deletion and the insertion of murine leukemia virus in the juxtamembrane region led to the production of abnormal transcripts starting from cryptic transcription start sites located halfway through the Notch1 gene and resulted in transcripts lacking most of the extracellular domain. As a result of these rearrangements, truncated Notch1 polypeptides resembling Notch1TM or Notch1IC were formed. In contrast, the 3' deletion led to the production of a C-terminal PEST motif-deleted transcript. The downstream target gene Hes1 was transcribed in a lymphoma with insertion of murine leukemia virus, but not in a lymphoma with a 5' deletion. These results indicate that in addition to Hes1 expression, other Notch1 pathway(s) have a role in thymic lymphomagenesis and suggest the presence of a novel mechanism for oncogenic activation of Notch1 by 5' deletion.

p27Kip1 (p27) is a cyclin-CDK inhibitor and negative regulator of cell proliferation. p27 also controls other cellular processes including migration and cytoplasmic p27 can act as an oncogene. Furthermore, cytoplasmic p27 promotes invasion and metastasis, in part by promoting epithelial to mesenchymal transition. Herein, we find that p27 promotes cell invasion by binding to and regulating the activity of Cortactin, a critical regulator of invadopodia formation. p27 localizes to invadopodia and limits their number and activity. p27 promotes the interaction of Cortactin with PAK1. In turn, PAK1 promotes invadopodia turnover by phosphorylating Cortactin, and expression of Cortactin mutants for PAK-targeted sites abolishes p27’s effect on invadopodia dynamics. Thus, in absence of p27, cells exhibit increased invadopodia stability due to impaired PAK1-Cortactin interaction, but their invasive capacity is reduced compared to wild-type cells. Overall, we find that p27 directly promotes cell invasion by facilitating invadopodia turnover via the Rac1/PAK1/Cortactin pathway.DOI: http://dx.doi.org/10.7554/eLife.22207.001

The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.