Intracellular Phospholipase A1 and Acyltransferase, Which Are Involved in Caenorhabditis elegans Stem Cell Divisions, Determine the sn-1 Fatty Acyl Chain of Phosphatidylinositol

Phosphatidylinositol (PI) is unique in the abundance of stearic acid at the sn-1 position. This fatty acid is thought to be incorporated through fatty acid remodeling. Here, we identified a phospholipase and acyltransferases involved in the fatty acid remodeling at the sn-1 position of PI and provide a link between the sn-1 fatty acid of PI and asymmetric cell division

An endomembrane zinc transporter negatively regulates systemic RNAi in Caenorhabditis elegans

Summary: Double-stranded RNA (dsRNA) regulates gene expression in a sequence-dependent manner. In Caenorhabditis elegans, dsRNA spreads through the body and leads to systemic RNA silencing. Although several genes involved in systemic RNAi have been genetically identified, molecules that mediate systemic RNAi remain largely unknown. Here, we identified ZIPT-9, a C. elegans homolog of ZIP9/SLC39A9, as a broad-spectrum negative regulator of systemic RNAi. We showed that RSD-3, SID-3, and SID-5 genetically act in parallel for efficient RNAi, and that zipt-9 mutants suppress the RNAi defects of all the mutants. Analysis of a complete set of deletion mutants for SLC30 and SLC39 family genes revealed that only zipt-9 mutants showed altered RNAi activity. Based on these results and our analysis using transgenic Zn2+ reporters, we propose that ZIPT-9-dependent Zn2+ homeostasis, rather than overall cytosolic Zn2+, modulates systemic RNAi activity. Our findings reveal a previously unknown function of zinc transporters in negative RNAi regulation

The Tumor Suppressor BCL7B Functions in the Wnt Signaling Pathway.

Human BCL7 gene family consists of BCL7A, BCL7B, and BCL7C. A number of clinical studies have reported that BCL7 family is involved in cancer incidence, progression, and development. Among them, BCL7B, located on chromosome 7q11.23, is one of the deleted genes in patients with Williams-Beuren syndrome. Although several studies have suggested that malignant diseases occurring in patients with Williams-Beuren syndrome are associated with aberrations in BCL7B, little is known regarding the function of this gene at the cellular level. In this study, we focused on bcl-7, which is the only homolog of BCL7 gene family in Caenorhabditis elegans, and analyzed bcl-7 deletion mutants. As a result, we found that bcl-7 is required for the asymmetric differentiation of epithelial seam cells, which have self-renewal properties as stem cells and divide asymmetrically through the WNT pathway. Distal tip cell development, which is regulated by the WNT pathway in Caenorhabditis elegans, was also affected in bcl-7-knockout mutants. Interestingly, bcl-7 mutants exhibited nuclear enlargement, reminiscent of the anaplastic features of malignant cells. Furthermore, in KATOIII human gastric cancer cells, BCL7B knockdown induced nuclear enlargement, promoted the multinuclei phenotype and suppressed cell death. In addition, this study showed that BCL7B negatively regulates the Wnt-signaling pathway and positively regulates the apoptotic pathway. Taken together, our data indicate that BCL7B/BCL-7 has some roles in maintaining the structure of nuclei and is involved in the modulation of multiple pathways, including Wnt and apoptosis. This study may implicate a risk of malignancies with BCL7B-deficiency, such as Williams-Beuren syndrome

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O‐mannosylation site on αα‐dystroglycan

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds β1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds β1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure

Knockout of <i>bcl-7</i> inhibits normal seam cell development in <i>Caenorhabditis elegans.</i>

<p><b>A</b>: Structure of <i>bcl-7</i>. Black boxes, exons; bent lines, introns. The region deleted in the strain <i>tm5268</i> is shown as a dotted line, below. The numbers indicate the location in the cosmid <i>C28H8</i>. <b>B–D</b>: Nomarski images of adult hermaphrodites. A wild-type hermaphrodite had complete alae (the region between arrows) (<b>B</b>); Alae were incomplete in <i>bcl-7</i> (<i>tm5268</i>) hermaphrodites. A region between dotted arrows indicates partial alae with only two ridges. The area without arrows indicates regions without alae (<b>C</b>); a transgenic rescue line carrying <i>Pbcl-7::bcl-7::egfp</i> reporters (<i>tm5268;tmEx2966</i>) with complete alae (three ridges) (<b>D</b>). <b>E–J</b>: Examples of SCM::GFP localization in wild-type, <i>tm5268</i>, and <i>tm5268;tmEx3496</i> hermaphrodites with <i>wIs51</i> (SCM::GFP). <i>tm5268;tmEx3496</i> is a transgenic rescue line carrying <i>Pbcl-7::bcl-7::mCherry</i> reporters. A wild-type L4 hermaphrodite expressing SCM::GFP in 16 seam cell nuclei (<b>E</b>), <i>tm5268</i> hermaphrodites carrying <i>scm::gfp</i> reporters (<b>H</b>–<b>G</b>), and a <i>tm5268;tmEx3496</i> hermaphrodite carrying <i>scm::gfp</i> reporters (<b>J</b>). Inserts show the Nomarski images of the same animals of the fluorescence images. <b>K</b>: Bar chart showing the average number of seam cells (about the worms at each larval stage, except for the middle L2 stage, and the young adult stage) or daughter cells (about the worms at middle L2 stage) expressing GFP in wild-type and <i>tm5268</i> hermaphrodites (n = 21–46). <b>L</b>: Percentages of worms with complete alae in wild-type, <i>tm5268</i>, and <i>tm5268;tmEx2966</i> adult hermaphrodites (n = 20–30). <b>M</b>: Bar chart showing the average number of seam cells expressing GFP in wild-type, <i>tm5268</i>, <i>tm5268;tmEx3496</i>, and <i>tm5268;tmEx4126[SCMp::bcl-7, SCMp::mCherry]</i> adult hermaphrodites with <i>wIs51</i> (SCM::GFP) (n = 15–23). Error bars indicate the standard error of the mean (SEM). Asterisks indicate statistical significance compared with each other. *p<0.05. **p<0.005. ***p<0.001. N.S.: no significance. Scale bar  = 25 µm.</p

Knockout of <i>bcl-7</i> affects gonadal development and germ cell proliferation in <i>Caenorhabditis elegans</i>.

<p><b>A–C</b>: A dissected adult germline of a wild-type adult hermaphrodite. A dissected gonad stained with DAPI (blue) and an anti-PH3 antibody (pink) as a mitotic cell-specific marker (<b>A</b>), and the regions of mitosis (<b>B</b>) and meiosis (<b>C</b>) of the gonad stained with DAPI. The arrowhead indicates a distal tip cell (DTC). The white arrows indicate the corresponding position of the gonad. <b>D–F</b>: A dissected germline of a <i>tm5268</i> adult hermaphrodite. A dissected gonad stained with DAPI (blue) and an anti-PH3 antibody (pink) as a mitotic cell-specific marker (D), and the regions of mitosis (<b>E</b>) and meiosis (<b>F</b>) of the gonad stained with DAPI. The arrowhead indicates a DTC. The white circles indicate the corresponding position of the gonad. <b>G–I</b>: A dissected germline of a <i>tm5268;tmEx2966</i> adult hermaphrodite. A dissected gonad stained with DAPI (blue) and an anti-PH3 antibody (pink) as a mitotic cell-specific marker (G), and the regions of mitosis (<b>H</b>) and meiosis (<b>I</b>) of the gonad stained with DAPI. The arrowhead indicates a DTC. The white rhomboid indicates the corresponding position of the gonad. <b>J–L</b>: A dissected germline of a <i>tm5268;tmEx3873</i> adult hermaphrodite carrying <i>Plag-2::bcl-7::egfp</i> as a DTC-specific rescue construct. A dissected gonad stained with DAPI (blue) (<b>J</b>), and the regions of mitosis (<b>K</b>) and meiosis (<b>L</b>) of the gonad stained with DAPI. The arrowhead indicates a DTC. The white squares indicate the corresponding position of the gonad. <b>M</b>: Bar chart indicating the brood size in wild-type, heterozygous and homozygous <i>bcl-7</i> mutants, and transgenic rescue lines. The extrachromosomal array <i>tmEx3875</i> contains <i>pTE5::bcl-7::egfp</i> as a germ cell-specific rescue construct, <i>tmEx4116</i> contains <i>lim-7::bcl-7::egfp</i> as a gonadal sheath cell-specific rescue construct, and <i>tmEx4121</i> contains both <i>Plag-2::bcl-7::egfp</i> and <i>lim-7::bcl-7::egfp</i>. Error bars indicate SEM. Asterisks indicate statistical significance. *p<0.05, **p<0.005, ***p<0.001. N.S.: no significance. Scale bar  = 25 µm.</p

Downregulation of <i>wrm-1</i> or <i>lsy-22</i> suppresses the phenotypes of <i>bcl-7</i> mutants.

<p><b>A–D</b>: Nomarski (<b>A, C</b>) and GFP images (<b>B, D</b>) of <i>tm5268</i> adult hermaphrodites carrying an <i>scm::gfp</i> reporter treated with empty vector (L4440) (<b>A, B</b>) or <i>wrm-1</i>-specific RNA (<b>C, D</b>). <b>E</b>: Bar chart representing the average number of seam cells in wild-type, <i>tm5268</i>, <i>tm5268</i>;L4440 (RNAi), or <i>tm5268</i>;<i>wrm-1</i> (RNAi) hermaphrodites at the L1, late L2, late L3, or young adult stages. <b>F–H</b>: Nomarski images of <i>tm5268</i> treated with L4440 (RNAi), <i>wrm-1</i> (RNAi), or <i>lsy-22</i> (RNAi). Eggs in the uterus are outlined with dotted lines (<b>G, H</b>). <b>I</b>: Percentages of the Ste phenotypes in wild-type and <i>tm5268</i> adult hermaphrodites treated with L4440 (RNAi), <i>wrm-1</i> (RNAi), or <i>lsy-22</i> (RNAi). Numbers of examined animals were more than 60 for all strains.Error bars indicate SEM. Asterisks indicate statistical significance. *p<0.05. **p<0.005. N.S.: no significance. Scale bar  = 50 µm.</p

BCL7B functions as a tumor suppressor via multiple pathways.

<p><b>A–D</b>: Nomarski (<b>A, C</b>) and DAPI (<b>B, D</b>) images of KATOIII cells transfected with control siRNA (<b>A, B</b>) and <i>BCL7B</i>-specific siRNA (<b>C, D</b>). <b>E–H</b>: Fluorescence images of KATOIII cells stained with WGA-Alexa Fluor 488 (<b>E, G</b>) and DAPI (<b>F, H</b>). These cells were transfected with control siRNA (<b>E, F</b>) or <i>BCL7B</i>-specific siRNA (<b>G, H</b>). The arrowheads indicate the presence of three nuclei in one cell. <b>I</b>: The percentages of the surviving KATOIII cells after transfection with control siRNA or <i>BCL7B</i>-specific siRNA and addition of actinomycin D. The number of living cells was counted 5 h after the addition of actinomycin D using trypan blue dye exclusion; this number was then divided by the total cell number. <b>J</b>: Percentages of KATOIII cells transfected with control siRNA or <i>BCL7B</i>-specific siRNA in the G₀/G₁, S, and G₂ phases. More than twenty thousand cells were counted for each analysis. <b>K, L</b>: The expression levels of ß-catenin (<b>K</b>) and c-FLIP (<b>L</b>) by the qRT-PCR analysis. <b>M</b>: Survival rates of KATOIII cells transfected with <i>EGFP</i>, as a control, or <i>BCL7B_EGFP</i>. The number of living cells was counted using trypan blue dye exclusion, and this number was then divided by the total cell number. <b>N</b>: The rates of apoptotic KATOIII cells transfected with <i>EGFP</i>, as a control, or <i>BCL7B_EGFP</i>. Apoptotic cells were stained for Annexin-V and/or 7-AAD and monitored by flow cytometric analysis. More than ten thousand cells were counted. Each experiment was performed three times independently. Error bars indicate SEM. The asterisks indicate the statistical significance of differences between the two groups. *p<0.05, **p<0.005, ***p<0.001. N.S.: no significance. Scale bar  = 25 µm.</p

BCL-7 is involved in the asymmetric localization of Wnt components in <i>Caenorhabditis elegans</i>.

<p><b>A–D</b>: Nomarski (<b>A, C</b>) and GFP (<b>B, D</b>) images of wild type (<b>A, B</b>) and <i>tm5268</i> (<b>C, D</b>) L2-stage hermaphrodites carrying a <i>wrm-1::gfp</i> reporter (<i>osIs5</i>) at pre-division states. Seam cells are traced with dotted ovals (<b>A, C</b>). <b>E–H</b>: Nomarski (<b>E, G</b>) and GFP (<b>F, H</b>) images showing the localization of WRM-1::GFP in two daughter cells of wild-type or <i>tm5268</i> L2 hermaphrodites at post-divisions. The pairs of daughter cells are outlined with dotted ovals. <b>I</b>: Frequency of the localization patterns of WRM-1::GFP. The equal and unequal signs indicate the relative intensities of nuclear WRM-1::GFP between two daughter cells. <b>J–M</b>: Nomarski (<b>J, L</b>) and GFP (<b>K, M</b>) images showing the localization of GFP::POP-1 (<i>qIs74</i>) in two daughter cells of wild-type (<b>J, K</b>) or <i>tm5268</i> (<b>L, M</b>) L1 hermaphrodites in post-division states. The pairs of daughter cells are outlined with dotted ovals. Asterisks indicate nuclei with stronger expression of GFP. <b>N</b>: The frequency of the localization patterns of GFP::POP-1. The equal and unequal signs indicate the relative intensities of nuclear GFP::POP-1 between two daughter cells. <b>O–R</b>: Nomarski (<b>O, Q</b>) and GFP (<b>P, R</b>) images showing the localization of GFP::POP-1 (<i>qIs74</i>) in SGPs of wild-type (<b>O, P</b>) or <i>tm5268</i> (<b>Q, R</b>) early L1 hermaphrodites. SGPs are outlined with dotted ovals. <b>S</b>: The frequency of the localization patterns of GFP::POP-1. The equal and unequal signs indicate the relative intensities of nuclear GFP::POP-1 between two daughter cells. Anterior is oriented toward the left, and ventral is oriented toward the bottom. <b>T</b>: Percentages of worms with the aberrant POP-1 asymmetry in both Z1 and Z4 cells, either Z1 or Z4 cells, and no cells in one worm between wild type and <i>bcl-7</i> mutant worms. Numbers of examined samples were more than 30 for all analyses. Scale bar  = 25 µm.</p