Muscle cell migrations of C. elegans are mediated by the alpha-integrin INA-1, Eph receptor VAB-1, and a novel peptidase homologue MNP-1

Cell migration is a fundamental process occurring during embryonic development and tissue morphogenesis. In the nematode Caenorhabditis elegans, morphogenesis of the body-wall musculature involves short-range migrations of 81 embryonic muscle cells from the lateral surface of the embryo towards the dorsal and ventral midlines. This study shows that mutations in ina-1 (α-integrin), as well as vab-1 (Eph receptor), and vab-2 (ephrin), display defects in embryonic muscle cell migration. Furthermore, an RNAi-based enhancer screen in an ina-1 weak loss-of-function background identified mnp-1 (matrix non-peptidase homologue-1) as a previously uncharacterized gene required for promoting proper migration of the embryonic muscle cells. mnp-1 encodes a membrane associated metalloproteinase homologue that is predicted to be catalytically inactive. Our data suggest that MNP-1 is expressed in migrating muscle cells and localizes to the plasma membrane with the non-peptidase domain exposed to the extra-cellular environment. Double-mutant analysis between mnp-1(RNAi), ina-1, and vab-1 mutations; as well as tissue specific rescue experiments; indicated that each of these gene products function predominantly independent of each other and from different cell types to affect muscle cell migration. Together these results suggest complex interactions between the adjacent epidermal, neuronal, and muscle cells are required to promote proper muscle cell migration during embryogenesis.     

The XLP syndrome protein SAP interacts with SH3 proteins to regulate T cell signaling and proliferation

The gene sap/shd1a, which encodes a 128-residue SH2 domain protein, is frequently deleted or mutated in the X-linked lymphoproliferative syndrome (XLP). The SAP SH2 domain differs from others in the same class in that it is not only capable of binding to a phosphotyrosine-containing peptide, it can also associate with an SH3 domain using a distinct surface. This novel mode of ligand-binding is initially discovered in the SLAM-SAP-Fyn complex that plays a critical role in T cell and natural killer cell activation. To identify additional binding partners for SAP, we screened a panel of 12 SH3 domains derived from regulatory proteins and identified NCK1 as a novel target of SAP in T cells. NMR analysis demonstrated that the NCK1 and Fyn SH3 domains possessed comparable affinities for SAP and engaged the same set of residues on the surface of the SAP SH2 domain. Depletion of SAP by siRNA caused a significant decrease in NCK1 tyrosine phosphorylation as well as the phosphorylation of other T cell receptor (TCR) downstream proteins such as LAT and SLP-76. Moreover, SAP was shown to regulate T cell proliferation through the MAP-kinase Erk. Taken together, our work identifies NCK1 as a novel physiological partner for SAP and a direct regulator of TCR signaling and T cell proliferation.     

UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.

The unc-5 gene is required for guiding pioneering axons and migrating cells along the body wall in C. elegans. In mutants, dorsal migrations are disrupted, but ventral and longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by transposon insertions. Based on genomic and cDNA sequencing, the gene encodes UNC-5, a transmembrane protein of 919 aa. The predicted extracellular N-terminus comprises two immunoglobulin and two thrombospondin type 1 domains. Except for an SH3-like motif, the large intracellular C-terminus is novel. Mosaic analysis shows that unc-5 acts in migrating cells and pioneering neurons. We propose that UNC-5 is a transmembrane receptor expressed on the surface of motile cells and growth cones to guide dorsal movements.     

RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.     

ced-4 and proto-oncogene tfg-1 antagonistically regulate cell size and apoptosis in C. elegans

BACKGROUND: Cell-size-control systems, coupled with apoptotic- and cell-proliferation-regulatory mechanisms, determine the overall dimensions of organs and organisms, and their dysregulation can lead to tumor formation. The interrelationship between cell-growth-regulatory mechanisms and apoptosis during normal development and cancer is not understood. The TRK-fused gene (TFG) promotes tumorigenesis when present in chromosomal rearrangements from various human-cancer types by unknown mechanisms. Apaf1/CED-4 is essential for apoptosis but has not been shown to function in cell-growth control. RESULTS: We found that loss of TFG-1, the TFG ortholog in Caenorhabditis elegans, results in supernumerary apoptotic corpses, whereas its overexpression is sufficient to inhibit developmentally programmed cell death. TFG-1 is also required for cells and nuclei to grow to normal size. Furthermore, we found that CED-4 is required for cell-growth inhibition in animals lacking TFG-1. However, caspases, the downstream effectors of CED-4-mediated apoptosis, are not required in TFG-1- or CED-4-regulated cell-size control. CED-4 acts to inhibit cell growth by antagonizing the effects of other conserved cell-size-regulating proteins, including cAMP response element binding (CREB) protein, translation-initiation factor eIF2B, and the nucleolar p53-interacting protein nucleostemin. CONCLUSIONS: These findings show that TFG-1 suppresses apoptosis and is essential for normal cell-size control, suggesting that abnormalities in the cell-growth-promoting and apoptosis-inhibiting functions of TFG might be responsible for its action in tumorigenesis. Also, they reveal that CED-4 plays a pivotal role in activating apoptosis and restricting cell and nuclear size, thereby determining the appropriate overall size of an animal. Thus, these findings reveal links between the control mechanisms for apoptosis and cell growth.     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.     

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc‐51‐like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a‐dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62‐positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient‐derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD‐associated p62 pathology.     

The C. elegans CBFbeta homologue BRO-1 interacts with the Runx factor, RNT-1, to promote stem cell proliferation and self-renewal

In this report, we investigate the C. elegans CBFbeta homologue, BRO-1. bro-1 mutants have a similar male-specific sensory ray loss phenotype to rnt-1 (the C. elegans homologue of the mammalian CBFbeta-interacting Runx factors), caused by failed cell divisions in the seam lineages. Our studies indicate that BRO-1 and RNT-1 form a cell proliferation-promoting complex, and that BRO-1 increases both the affinity and specificity of RNT-1-DNA interactions. Overexpression of bro-1, like rnt-1, leads to an expansion of seam cell number and co-overexpression of bro-1 and rnt-1 results in massive seam cell hyperplasia. Finally, we find that BRO-1 appears to act independently of RNT-1 in certain situations. These studies provide new insights into the function and regulation of this important cancer-associated DNA-binding complex in stem cells and support the view that Runx/CBFbeta factors have oncogenic potential.     

The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans

In metazoans, microRNAs (miRNAs) carry out various regulatory functions through association with multiprotein miRNA-induced silencing complexes (miRISCs) that contain Dicer and Argonaute proteins. How miRNAs regulate the expression of their mRNA targets remains a major research question. We have identified the C. elegans ain-1 gene through a genetic suppressor screen and shown that it functions with the heterochronic genetic pathway that regulates developmental timing. Biochemical analysis indicates that AIN-1 interacts with protein complexes containing an Argonaute protein, Dicer, and miRNAs. AIN-1 shares homology with the candidate human neurological disease protein GW182, shown to localize in cytoplasmic processing bodies that are sites of mRNA degradation and storage. A functional AIN-1::GFP also localizes at the likely worm processing bodies. When coexpressed from transgenes, AIN-1 targets ALG-1 to the foci. These results suggest a model where AIN-1 regulates a subset of miRISCs by localization to the processing bodies, facilitating degradation or translational inhibition of mRNA targets.     

Down syndrome candidate region-1 protein interacts with Tollip and positively modulates interleukin-1 receptor-mediated signaling

Background

The Down syndrome candidate region-1 gene (DSCR1, also known as RCAN1) is situated close to the Down Syndrome Critical Region (DSCR), which contains genes responsible for many features of Down syndrome. DSCR1 modulates calcineurin phosphatase activity, though its functional role is incompletely understood.

Methods

Here we investigated the role of DSCR1-1S isoform in IL-1 receptor (IL-1R)-mediated signaling by analyzing interaction between DSCR1-1S and the IL-1R pathway components Tollip, IRAK-1, and TRAF6.

Results

Co-immunoprecipitation analyses of HEK293 cells revealed that DSCR1-1S interacted with Tollip, an IRAK-1 inhibitor, leading to the dissociation of IRAK-1 from Tollip. Similarly, both DSCR1-1S and Tollip interacted with TRAF6, with DSCR1 reducing interaction between Tollip and TRAF6. DSCR1-1S also stimulated IL-1R-mediated signaling pathways, TAK1 activation, NF-κB transactivation, and IL-8 production, all downstream consequences of IL-1R activation.

General significance

Together, these results suggest that DSCR1-1S isoform positively modulates IL-1R-mediated signaling pathways by regulating Tollip/IRAK-1/TRAF6 complex formation.     

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.     

The cell signaling adaptor protein EPS-8 is essential for C. elegans epidermal elongation and interacts with the ankyrin repeat protein VAB-19

Background

The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described.

Methodology/Principal Findings

In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 null mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures.

Conclusions/Significance

EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.     

Plant Bax Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death

Autophagy is an evolutionarily conserved catabolic process and is involved in the regulation of programmed cell death during the plant immune response. However, mechanisms regulating autophagy and cell death are incompletely understood. Here, we demonstrate that plant Bax inhibitor-1 (BI-1), a highly conserved cell death regulator, interacts with ATG6, a core autophagy-related protein. Silencing of BI-1 reduced the autophagic activity induced by both N gene-mediated resistance to Tobacco mosaic virus (TMV) and methyl viologen (MV), and enhanced N gene-mediated cell death. In contrast, overexpression of plant BI-1 increased autophagic activity and surprisingly caused autophagy-dependent cell death. These results suggest that plant BI-1 has both prosurvival and prodeath effects in different physiological contexts and both depend on autophagic activity.     

Wnt signaling and a Hox protein cooperatively regulate psa-3/Meis to determine daughter cell fate after asymmetric cell division in C. elegans

Asymmetric cell division is a mechanism for achieving cellular diversity. In C. elegans, many asymmetric cell divisions are controlled by the Wnt-MAPK pathway through POP-1/TCF. It is poorly understood, however, how POP-1 determines the specific fates of daughter cells. We found that nob-1/Hox, ceh-20/Pbx, and a Meis-related gene, psa-3, are required for asymmetric division of the T hypodermal cell. psa-3 expression was asymmetric between the T cell daughters, and it was regulated by POP-1 through a POP-1 binding site in the psa-3 gene. psa-3 expression was also regulated by NOB-1 and CEH-20 through a NOB-1 binding sequence in a psa-3 intron. PSA-3 can bind CEH-20 and function after the T cell division to promote the proper fate of the daughter cell. These results indicate that cooperation between Wnt signaling and a Hox protein functions to determine the specific fate of a daughter cell.     

C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are well-conserved RNA-binding proteins, which regulate mRNA translation mainly through control of poly(A) elongation. Here, we show that CPB-3, one of the four CPEB homologs in C. elegans, positively regulates multiple aspects of oocyte production. CPB-3 protein was highly expressed in early meiotic regions of the hermaphrodite gonad. Worms deficient in cpb-3 were apparently impaired in germ cell proliferation and differentiation including sperm/oocyte switching and progression of female meiosis. We also show that cpb-3 is likely to promote the meiotic entry in parallel with gld-3, a component of one of the redundant but essential genetic pathways for the entry to and progression through meiosis. Taken together, CPEB appears to have a conserved role in the early phase of meiosis and in the sperm/oocyte specification, in addition to its reported function during meiotic progression.     

Autophagy genes coordinate with the class II PI/PtdIns 3-kinase PIKI-1 to regulate apoptotic cell clearance in C. elegans

Phagocytosis and autophagy are two lysosome-mediated cellular degradation pathways designed to eliminate extracellular and intracellular constituents, respectively. Recent studies suggest that these two processes intersect. Several autophagy proteins have been shown to participate in clearance of apoptotic cells, but whether and how the autophagy pathway is involved is unclear. Here we showed that loss of function mutations in 19 genes acting at overlapping or distinct stages of autophagy caused increased numbers of cell corpses in C. elegans embryos. In contrast, genes that mediate specific clearance of P granules or protein aggregates through autophagy are dispensable for cell corpse removal. We showed that defective autophagy impairs phagosome maturation and that autophagy genes act in parallel to the class II phosphoinositide (PI)/phosphatidylinositol (PtdIns) 3-kinase PIKI-1 to regulate phagosomal PtdIns3P in a similar manner as VPS-34. Our data indicate that autophagy may coordinate with PIKI-1 to promote phagosome maturation, thus ensuring efficient clearance of apoptotic cells.     

The bromodomain protein LEX-1 acts with TAM-1 to modulate gene expression in C. elegans

In many organisms, repetitive DNA serves as a trigger for gene silencing. However, some gene expression is observed from repetitive genomic regions such as heterochromatin, suggesting mechanisms exist to modulate the silencing effects. From a genetic screen in C. elegans, we have identified mutations in two genes important for expression of repetitive sequences: lex-1 and tam-1. Here we show that lex-1 encodes a protein containing an ATPase domain and a bromodomain. LEX-1 is similar to the yeast Yta7 protein, which maintains boundaries between silenced and active chromatin. tam-1 has previously been shown to encode a RING finger/B-box protein that modulates gene expression from repetitive DNA. We find that lex-1, like tam-1, acts as a class B synthetic multivulva (synMuv) gene. However, since lex-1 and tam-1 mutants have normal P granule localization, it suggests they act through a mechanism distinct from other class B synMuvs. We observe intragenic (interallelic) complementation with lex-1 and a genetic interaction between lex-1 and tam-1, data consistent with the idea that the gene products function in the same biological process, perhaps as part of a protein complex. We propose that LEX-1 and TAM-1 function together to influence chromatin structure and to promote expression from repetitive sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.