Phospholipase C-ε Regulates Epidermal Morphogenesis in Caenorhabditis elegans

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP3)-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP3 production is stimulated is unknown. IP3 is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-ε produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP3 receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-γ and EGL-8/PLC-β can compensate for reduced PLC-1 activity. Our work places PLC-ε into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-ε

Visualization of C. elegans transgenic arrays by GFP

BACKGROUND: Targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific DNA sequence, the lac operator (lacO), allows visualization of chromosomes in yeast and mammalian cells. In principle this method of visualization could be used for genetic mosaic analysis, which requires cell-autonomous markers that can be scored easily and at single cell resolution. The C. elegans lin-3 gene encodes an epidermal growth factor family (EGF) growth factor. lin-3 is expressed in the gonadal anchor cell and acts through LET-23 (transmembrane protein tyrosine kinase and ortholog of EGF receptor) to signal the vulval precursor cells to generate vulval tissue. lin-3 is expressed in the vulval cells later, and recent evidence raises the possibility that lin-3 acts in the vulval cells as a relay signal during vulval induction. It is thus of interest to test the site of action of lin-3 by mosaic analysis. RESULTS: We visualized transgenes in living C. elegans by targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific 256 sequence repeat of the lac operator (lacO) incorporated into transgenes. We engineered animals to express a nuclear-localized GFP-LacI fusion protein. C. elegans cells having a lacO transgene result in nuclear-localized bright spots (i.e., GFP-LacI bound to lacO). Cells with diffuse nuclear fluorescence correspond to unbound nuclear localized GFP-LacI. We detected chromosomes in living animals by chromosomally integrating the array of the lacO repeat sequence and visualizing the integrated transgene with GFP-LacI. This detection system can be applied to determine polyploidy as well as investigating chromosome segregation. To assess the GFP-LacI•lacO system as a marker for mosaic analysis, we conducted genetic mosaic analysis of the epidermal growth factor lin-3, expressed in the anchor cell. We establish that lin-3 acts in the anchor cell to induce vulva development, demonstrating this method's utility in detecting the presence of a transgene. CONCLUSION: The GFP-LacI•lacO transgene detection system works in C. elegans for visualization of chromosomes and extrachromosomal transgenes. It can be used as a marker for genetic mosaic analysis. The lacO repeat sequence as an extrachromosomal array becomes a valuable technique allowing rapid, accurate determination of spontaneous loss of the array, thereby allowing high-resolution mosaic analysis. The lin-3 gene is required in the anchor cell to induce the epidermal vulval precursors cells to undergo vulval development

Detailed Regulatory Mechanism of the Interaction between ZO-1 PDZ2 and Connexin43 Revealed by MD Simulations

The gap junction protein connexin43 (Cx43) binds to the second PDZ domain of Zonula occludens-1 (ZO-1) through its C-terminal tail, mediating the regulation of gap junction plaque size and dynamics. Biochemical study demonstrated that the very C-terminal 12 residues of Cx43 are necessary and sufficient for ZO-1 PDZ2 binding and phosphorylation at residues Ser (-9) and Ser (-10) of the peptide can disrupt the association. However, only a crystal structure of ZO-1 PDZ2 in complex with a shorter 9 aa peptide of connexin43 was solved experimentally. Here, the interactions between ZO-1 PDZ2 and the short, long and phosphorylated Cx43 peptides were studied using molecular dynamics (MD) simulations and free energy calculation. The short peptide bound to PDZ2 exhibits large structural variations, while the extension of three upstream residues stabilizes the peptide conformation and enhanced the interaction. Phosphorylation at Ser(-9) significantly weakens the binding and results in conformational flexibility of the peptide. Glu210 of ZO-1 PDZ2 was found to be a key regulatory point in Cx43 binding and phosphorylation induced dissociation

sel-11 and cdc-42, Two Negative Modulators of LIN-12/Notch Activity in C. elegans

Background: LIN-12/Notch signaling is important for cell-cell interactions during development, and mutations resulting in constitutive LIN-12/Notch signaling can cause cancer. Loss of negative regulators of lin-12/Notch activity has the potential for influencing cell fate decisions during development and the genesis or aggressiveness of cancer. Methodology/Principal Findings: We describe two negative modulators of lin-12 activity in C. elegans. One gene, sel-11, was initially defined as a suppressor of a lin-12 hypomorphic allele; the other gene, cdc-42, is a well-studied Rho GTPase. Here, we show that SEL-11 corresponds to yeast Hrd1p and mammalian Synoviolin. We also show that cdc-42 has the genetic properties consistent with negative regulation of lin-12 activity during vulval precursor cell fate specification. Conclusions/Significance: Our results underscore the multiplicity of negative regulatory mechanisms that impact on lin-12/ Notch activity and suggest novel mechanisms by which constitutive lin-12/Notch activity might be exacerbated in cancer

Identification of Novel Targets of CSL-Dependent Notch Signaling in Hematopoiesis

Somatic activating mutations in the Notch1 receptor result in the overexpression of activated Notch1, which can be tumorigenic. The goal of this study is to understand the molecular mechanisms underlying the phenotypic changes caused by the overexpression of ligand independent Notch 1 by using a tetracycline inducible promoter in an in vitro embryonic stem (ES) cells/OP9 stromal cells coculture system, recapitulating normal hematopoiesis. First, an in silico analysis of the promoters of Notch regulated genes (previously determined by microarray analysis) revealed that the motifs recognized by regulatory proteins known to mediate hematopoiesis were overrepresented. Notch 1 does not bind DNA but instead binds the CSL transcription factor to regulate gene expression. The in silico analysis also showed that there were putative CSL binding sites observed in the promoters of 28 out of 148 genes. A custom ChIP-chip array was used to assess the occupancy of CSL in the promoter regions of the Notch1 regulated genes in vivo and showed that 61 genes were bound by activated Notch responsive CSL. Then, comprehensive mapping of the CSL binding sites genome-wide using ChIP-seq analysis revealed that over 10,000 genes were bound within 10 kb of the TSS (transcription start site). The majority of the targets discovered by ChIP-seq belong to pathways that have been shown by others to crosstalk with Notch signaling. Finally, 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of in vitro hematopoiesis. Thirty one miRNA were up-regulated and fifty two were down-regulated. Overexpression of Notch1 altered this pattern of expression of microRNA: six miRNAs were up-regulated and four were down regulated as a result of activated Notch1 overexpression during the course of hematopoiesis. Time course analysis of hematopoietic development revealed that cells with Notch 1 overexpression mimic miRNA expression of cells in a less mature stage, which is consistent with our previous biological characterization

Tight junctions: from simple barriers to multifunctional molecular gates

Epithelia and endothelia separate different tissue compartments and protect multicellular organisms from the outside world. This requires the formation of tight junctions, selective gates that control paracellular diffusion of ions and solutes. Tight junctions also form the border between the apical and basolateral plasma-membrane domains and are linked to the machinery that controls apicobasal polarization. Additionally, signalling networks that guide diverse cell behaviours and functions are connected to tight junctions, transmitting information to and from the cytoskeleton, nucleus and different cell adhesion complexes. Recent advances have broadened our understanding of the molecular architecture and cellular functions of tight junctions

TM4SF10 gene sequencing in XLMR patients identifies common polymorphisms but no disease-associated mutation

BACKGROUND: The TM4SF10 gene encodes a putative four-transmembrane domains protein of unknown function termed Brain Cell Membrane Protein 1 (BCMP1), and is abundantly expressed in the brain. This gene is located on the short arm of human chromosome X at p21.1. The hypothesis that mutations in the TM4SF10 gene are associated with impaired brain function was investigated by sequencing the gene in individuals with hereditary X-linked mental retardation (XLMR). METHODS: The coding region (543 bp) of TM4SF10, including intronic junctions, and the long 3' untranslated region (3 233 bp), that has been conserved during evolution, were sequenced in 16 male XLMR patients from 14 unrelated families with definite, or suggestive, linkage to the TM4SF10 gene locus, and in 5 normal males. RESULTS: Five sequence changes were identified but none was found to be associated with the disease. Two of these changes correspond to previously known SNPs, while three other were novel SNPs in the TM4SF10 gene. CONCLUSION: We have investigated the majority of the known MRX families linked to the TM4SF10 gene region. In the absence of mutations detected, our study indicates that alterations of TM4SF10 are not a frequent cause of XLMR.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.SCOPUS: ar.jinfo:eu-repo/semantics/publishe