|Beta-catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions.|
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|PMID: 18497747 Owner: NLM Status: MEDLINE|
|Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/beta-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as beta-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A(1) (PLA(1)), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of beta-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA(1) gene, cortical beta-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of beta-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of beta-catenin.|
|Takahiro Kanamori; Takao Inoue; Taro Sakamoto; Keiko Gengyo-Ando; Masafumi Tsujimoto; Shohei Mitani; Hitoshi Sawa; Junken Aoki; Hiroyuki Arai|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2008-05-22|
|Title: The EMBO journal Volume: 27 ISSN: 1460-2075 ISO Abbreviation: EMBO J. Publication Date: 2008 Jun|
|Created Date: 2008-06-18 Completed Date: 2008-07-15 Revised Date: 2013-06-05|
Medline Journal Info:
|Nlm Unique ID: 8208664 Medline TA: EMBO J Country: England|
|Languages: eng Pagination: 1647-57 Citation Subset: IM|
|Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.|
|APA/MLA Format Download EndNote Download BibTex|
Caenorhabditis elegans / cytology*, genetics, metabolism*
Caenorhabditis elegans Proteins / metabolism*
Cytoskeletal Proteins / metabolism
Guanine Nucleotide Exchange Factors
Mitotic Spindle Apparatus / metabolism
Mutant Proteins / isolation & purification, metabolism
Mutation / genetics
Phospholipases A1 / metabolism*
Stem Cells / cytology*
Subcellular Fractions / metabolism
Vulva / cytology
beta Catenin / metabolism*
rab GTP-Binding Proteins / metabolism
|0/Caenorhabditis elegans Proteins; 0/Cytoskeletal Proteins; 0/Guanine Nucleotide Exchange Factors; 0/Mutant Proteins; 0/WRM-1 protein, C elegans; 0/beta Catenin; EC 126.96.36.199/Phospholipases A1; EC 3.6.1.-/rab GTP-Binding Proteins|
Journal ID (nlm-ta): EMBO J
Publisher: Nature Publishing Group
Copyright © 2008, European Molecular Biology Organization
open-access: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.
Received Day: 10 Month: 01 Year: 2008
Accepted Day: 28 Month: 04 Year: 2008
Print publication date: Day: 18 Month: 06 Year: 2008
Electronic publication date: Day: 22 Month: 05 Year: 2008
Volume: 27 Issue: 12
First Page: 1647 Last Page: 1657
Publisher Item Identifier: emboj2008102
PubMed Id: 18497747
|β-Catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions Alternate Title:Phospholipase A1 controls asymmetric divisions|
1Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
2Laboratory of Cellular Biochemistry, RIKEN, Saitama, Japan
3CREST, Japan Science and Technology Agency, Saitama, Japan
4School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan
5Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan
6Laboratory for Cell Fate Decision, RIKEN Center for Developmental Biology, Kobe, Japan
7Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan
8Department of Molecular & Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan
9PRESTO, Japan Science and Technology Agency, Saitama, Japan
|aGraduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel: +81 3 5841 4720; Fax: +81 3 3818 3173; E-mail: firstname.lastname@example.org
Asymmetric division is an attractive means for stem cells to balance self-renewal and differentiation (Morrison and Kimble, 2006). To divide asymmetrically, stem cells become polarized prior to asymmetric divisions; Drosophila neural stem cells, for example, establish an axis of polarity to localize cell-fate determinants to one side of the cells and to orient the mitotic spindle correctly (Betschinger and Knoblich, 2004; Yu et al, 2006).
In Caenorhabditis elegans, the polarity of the most asymmetric divisions is regulated by the Wnt signalling pathway, dubbed as the ‘Wnt/β-catenin asymmetry pathway' (Thorpe et al, 2000; Herman, 2002; Mizumoto and Sawa, 2007b). In the canonical Wnt pathway, binding of Wnt to its receptor, Frizzled (Fz), results in activation of Dishevelled (Dsh), which in turn inactivates the β-catenin destruction complex, leading to β-catenin stabilization. The stabilized β-catenin enters the nucleus, where it interacts with TCF transcription factors and converts them from repressors to activators (Logan and Nusse, 2004; Clevers, 2006). In the Wnt/β-catenin asymmetry pathway, Wnt signalling components become polarized in the mother cells by Wnt proteins, and then polarized Wnt signalling produces two daughter cells with distinct transcriptional activities of TCF pop-1. Model asymmetric divisions for the Wnt/β-catenin asymmetry pathway include that of the T blast cell. Before T cell division, the Wnt protein lin-44, which is expressed posterior to the T cell, induces asymmetric cortical localizations of the canonical Wnt pathway components; Fz lin-17, Dsh dsh-2 and Dsh mig-5 localize to the posterior side of the T cell, and β-catenin wrm-1 and some of the destruction complex proteins localize to the anterior cortex (Takeshita and Sawa, 2005; Goldstein et al, 2006; Mizumoto and Sawa, 2007a). In telophase, WRM-1 accumulates into the posterior nucleus, where it appears to promote the nuclear export of POP-1 (Lo et al, 2004), and the nuclear level of POP-1 becomes asymmetric between two daughter cells (POP-1 asymmetry). Recently, it was shown that another β-catenin, SYS-1 acts as a coactivator for POP-1 (Kidd et al, 2005), and that in many A–P divisions, the nuclear localization of SYS-1 is also asymmetric in a manner reciprocal to POP-1 asymmetry (Huang et al, 2007; Phillips et al, 2007). This reciprocal asymmetry of POP-1 and SYS-1 provides a robust change in POP-1 transcriptional activity and seems to be a universal mechanism for cell-fate specification during animal development (Huang et al, 2007; Phillips et al, 2007). Among the cells that undergo asymmetric divisions during C. elegans development, the lateral epithelial seam cells are unique in that they have characteristics of stem cells. First, seam cells repeatedly divide to generate one daughter with a seam cell fate (self-renewal) and one daughter that fuses with the hypodermal syncytium (differentiation). Second, like many stem cells in other organisms, seam cells can also divide symmetrically to expand in number during development. Therefore, seam cells can provide a good model for the study of stem cells (Mizumoto and Sawa, 2007b).
Phospholipids, in addition to acting as structural components of cell membrane, also regulate many biological processes by acting as lipid mediators, second messengers and subcellular microenvironment. In many cases, these functions are mediated by a diverse group of phospholipases (PLs) that are classified into four groups (PLA, PLB, PLC and PLD) according to the bond hydrolysed on phospholipid substrates. PLA is represented by the two isoenzymes PLA1 and PLA2 that differ in the fatty acid they remove from a glyceride; PLA1 hydrolyses sn-1 fatty acids attached to phospholipids to produce 2-acyl-lysophospholipids, whereas PLA2 hydrolyses sn-2 fatty acids. PLA2 enzymes, the largest group of PLs, have essential functions in the generation of lipid mediators such as prostaglandins and leukotrienes. In contrast, the molecular identity of PLA1 enzymes was not clarified until recently (Higgs et al, 1998). In mammals, at least three members of the intracellular PLA1 family exist, namely PA-PLA1, KIAA0725 and p125, none of which show sequence homology with other known PLs (Inoue and Aoki, 2006). Intracellular PLA1 is highly conserved in a wide range of eukaryotic organisms from yeast, plants to mammals. However, most of their physiological functions remain to be elucidated. Our laboratory is currently interested in studying the intracellular PLA1 family. Using a reverse genetic approach, we show here an intracellular PLA1, ipla-1, as a regulator of asymmetric divisions in C. elegans. Disruption of the ipla-1 gene causes defects in spindle orientation, asymmetric cell-fate determination and asymmetric cortical localization of WRM-1 in the terminal asymmetric divisions of seam cells. We have also used a forward genetic approach to identify genes that functionally interact with ipla-1 in the asymmetric divisions, and provide evidence to suggest that ipla-1 regulates membrane trafficking to control cortical asymmetry of WRM-1 during the terminal asymmetric divisions of seam cells.
Our database searches identified one intracellular PLA1 family member, which we named ipla-1, in C. elegans. ipla-1 is predicted to encode a protein of 840 amino acids, including a serine esterase consensus sequence motif (GxSxG) and the DDHD domain, and shows 31% identity to human PA-PLA1 (Supplementary Figure 1B and C).
To investigate the functional roles of ipla-1, we isolated two ipla-1 deletion alleles, designated ipla-1(xh13) and ipla-1(tm471), by PCR-based screening of UV-TMP-mutagenized libraries. The xh13 allele deleted 1138 bp encoding exons 6 and 7, whereas tm471 harboured a 1172 bp deletion at exon 8 (Supplementary Figure 1A). None of the two alleles of ipla-1 showed detectable IPLA-1 protein by western blot, suggesting that they are strong loss-of-function or null alleles (Supplementary Figure 1D). These two ipla-1 mutants were viable and fertile; however, some of ipla-1 mutants exhibited vulval defects, including a protruding vulva and occasional vulval bursting (Supplementary Figure 2A and B). Because xh13 and tm471 were phenotypically indistinguishable in the initial characterization, we used ipla-1(xh13) mainly in subsequent analyses.
The vulva is formed from the descendants of three vulval precursor cells (VPCs). The fates of the VPCs are specified by an inductive signal from the gonadal anchor cell, which appears to be controlled by an inhibitory signal from the major hypodermal syncytium (hyp7) (Sternberg, 2005). In addition, recent studies have suggested that maintenance of seam cells is important for structural integrity of the vulva (Pellis-van Berkel et al, 2005; Smith et al, 2005). To identify the cells in which ipla-1 is required for proper morphology of the vulva, we expressed an ipla-1 minigene in subsets of these cells using specific promoters. Proper vulval morphology was restored to ipla-1 mutants by expression of ipla-1 with the dpy-7 promoter, which drives expression in hyp7 and weakly in seam cells (Gilleard et al, 1997), and with the seam cell-specific scm promoter (Koh and Rothman, 2001). In contrast, expression of ipla-1, under the control of either the hyp7-specific egl-15 enhancer elements (Huang and Stern, 2004) or the VPC-specific lin-31 promoter (Tan et al, 1998) failed to rescue the vulval defects (Supplementary Figure 2C). These results suggest that defects in seam cells cause abnormal vulval morphogenesis in ipla-1 mutants.
The lateral seam cells are specialized epithelial cells. During each larval stage, seam cells divide asymmetrically in a stem cell-like manner producing an anterior daughter cell that fuses with hyp7 and loses the expression of the seam cell marker (scm::gfp, transgene wIs51), and a posterior daughter cell that assumes the seam cell fate again and continues to express scm::gfp (Figure 2A and D).
To understand the nature of seam cell defects in ipla-1 mutants, we first analysed the number of seam cells using scm::gfp. Wild-type adult hermaphrodites usually contain evenly spaced 16 scm::gfp-positive nuclei on each side of the animals, derived from the 10 embryonically derived blast cells H0, H1, H2, V1-6 and T (Figures 1A and 2A). ipla-1 mutants, by contrast, contained more than 16 unevenly spaced seam cell nuclei (Figure 1B–D; Table I, see group A), some of which lay outside a row of seam cells (Figure 1C, arrow), and some of which lay close to each other (Figure 1C, arrowhead). Furthermore, scm::gfp-positive nuclei were occasionally lost (Figure 1D, arrowheads), and in such area seam cells were separated by gaps as indicated by a break in ajm-1::gfp, cdh-3::gfp and lateral alae (Supplementary Figure 3). To determine the cause of the missing or additional seam cells in ipla-1 mutants, we then followed the development of seam cells during each larval stage. Although ipla-1 mutants showed normal number and alignment of seam cells during the L2 and L3 stages, they exhibited aberrant number and alignment of seam cells at the L4 stage (Figure 1E). This suggests that ipla-1 expression is required for the terminal asymmetric divisions of seam cells that occur at the beginning of the L4 stage, which we call hereafter the ‘S4 divisions' (Seam cell divisions at the L4 stage). To confirm this idea, we expressed ipla-1 cDNA by the heat-shock promoter at specific developmental stages. We found that heat-shock treatments before but not after the S4 divisions (even at the end of the L3 stage) restored proper number and alignment of seam cells to ipla-1 mutants (Figure 1F). Thus, presence of IPLA-1 protein during the S4 divisions is sufficient for proper number and alignment of seam cells at the adult stage.
We next conducted cell-specific rescue experiments to confirm the cell autonomy of ipla-1 function. The seam cell defects were fully rescued by expression of ipla-1 under the dpy-7 promoter and the seam cell-specific scm promoter. No rescue was obtained when ipla-1 was expressed with the hyp7-specific egl-15 enhancer elements (Table I, see group A). Furthermore, expression of catalytically inactive mutant IPLA-1 (ipla-1 S489A, see Supplementary data, Supplementary Figure 1A and C, for details) by the scm promoter did not rescue the seam cell defects of ipla-1 mutants (Table I, see group A). These results indicate that ipla-1 functions cell-autonomously in seam cells to regulate their S4 divisions through its enzymatic activity.
We next analysed seam cells undergoing the S4 divisions and performed lineage analyses. In wild-type animals, the S4 divisions invariably occurred parallel to the anterior–posterior axis (A–P axis) (Figure 2B). In ipla-1 mutants, however, orientation of the S4 divisions was essentially randomized relative to the A–P axis (asterisks in Figure 2C). To quantify this defect, we measured the angle between a line connecting the two daughter nuclei and the A–P axis as depicted in Figure 2F (see Supplementary data). In wild-type animals, the measured angle was always less than 10° (n=110; Figure 2G). In ipla-1 mutants, the majority of seam cells underwent the S4 divisions in various directions (52%, n=107; Figure 2H). These results suggest that ipla-1 mutants have defects in spindle orientation during the S4 divisions. Furthermore, detailed lineage analyses revealed that in ipla-1 mutants, the asymmetry of the divisions was often disrupted (62%, n=53), leading to the transformation of the anterior daughters from hyp7 cells to seam cells or to the reversal of these cell fates (Figure 2E and I). It is noted that seam cells that divided normally in the A–P direction did have defects in the asymmetry of the divisions (Figure 2I; types I and II), indicating that defects in cell-fate determination are not secondary to the spindle orientation phenotype. We conclude that ipla-1 mutants are defective in both spindle orientation and cell-fate determination in the S4 divisions.
In C. elegans, many asymmetric divisions are controlled by the Wnt/β-catenin asymmetry pathway, including β-catenin wrm-1 and TCF pop-1 transcription factor (Mizumoto and Sawa, 2007b). In several asymmetric divisions, including those of seam cells at the L1 stage, WRM-1 localizes asymmetrically to the anterior cortex in mother cells (Nakamura et al, 2005; Takeshita and Sawa, 2005; Mizumoto and Sawa, 2007a). After the asymmetric divisions, the anterior daughters have more nuclear GFP::POP-1 than posterior daughters. This phenomenon has been dubbed ‘POP-1 asymmetry' (Lin et al, 1998).
To test whether ipla-1 mutation influences the Wnt/β-catenin asymmetry pathway in the S4 divisions, we determined the subcellular localization of WRM-1::GFP and GFP::POP-1. In wild-type animals, WRM-1::GFP always localized to the anterior cortex in seam cells before the S4 divisions (n=21; Figure 3A, B and D). However, the cortical localization of WRM-1 was randomized in ipla-1 mutants; WRM-1::GFP was symmetrically localized (22%), absent from the cortex (34%) and occasionally enriched posteriorly (6%) (n=34; Figure 3C and D). We also found that ipla-1 mutants had defects in the POP-1 asymmetry just after the S4 divisions; the levels of POP-1 were equally high in the two daughters (32%), and higher in the posterior daughters (36%) in ipla-1 mutants (n=25; Figure 3G and H), whereas the level of GFP::POP-1 was always higher in the anterior daughters in wild-type animals (n=19; Figure 3E, F and H). The frequencies of these mislocalization phenotypes were similar to that of defects in cell-fate determination after the S4 divisions in ipla-1 mutants (62%; Figure 2I). These results indicate that ipla-1 is required for the formation and/or maintenance of cortical asymmetry of WRM-1 prior to the S4 divisions.
To understand the molecular mechanism underlying the regulation of cortical WRM-1 asymmetry mediated by ipla-1, we carried out genetic screens for mutations that suppress the seam cell defects of ipla-1 mutants (see Supplementary data). In a screen of 3000 haploid mutagenized ipla-1(xh13);wIs51 genomes, we isolated two strong suppressor mutations. The two recessive suppressor alleles, xh22 and xh23, mapped to chromosome IV and did complement each other, indicating that they identify two distinct loci. On their own, these suppressor alleles displayed wild-type seam cell number and alignment and had no obvious morphological defects (Table I, see groups B and C; and data not shown). However, they effectively suppressed the seam cell phenotype of ipla-1 mutants with almost complete penetrance (Table I, see groups B and C). This suppression was not simply due to cell cycle arrest or reducing seam cell number before the S4 divisions, because evenly spaced seam cells divided asymmetrically and parallel to the A–P axis in both sup and ipla-1;sup double mutants during the S4 divisions (Figure 4F and data not shown; sup refers to any suppressor mutation). Notably, both ipla-1; xh22 and ipla-1; xh23 double mutants had normal vulvae, supporting the notion that abnormal vulval morphology is caused by the seam cell defects in ipla-1 mutants (Supplementary Figure 2 and data not shown).
We mapped xh22 and xh23 to 0.5-cM intervals on the right arm of chromosome IV. Of the genes predicted to reside in each region, inactivation of mon-2 and tbc-3 by RNAi gave rise to suppression of ipla-1 seam cell defects (Table I, see groups B and C; see Supplementary data). We subsequently found by germline transformation experiments that mon-2 and tbc-3 genes can rescue the suppression phenotype in ipla-1;xh22 and ipla-1;xh23, respectively (see Table I groups B and C, and below for rescue by mCherry fusion genes).
mon-2 encodes a homologue of the yeast Mon2p, which is highly conserved through evolution and is related to the GBF and BIG family ADP ribosylation factor (Arf) guanine nucleotide exchange factors (GEFs) (D'Souza-Schorey and Chavrier, 2006) over much of its length but lacks the catalytic domain (Figure 4E) (Efe et al, 2005; Gillingham et al, 2006). Sequencing of the mon-2 gene in xh22 mutants identified a nonsense mutation in the middle of the protein (Trp787Stop) (Figure 4D and E). This mutation would result in loss of the C-terminal region that is reported to be essential for yeast Mon2p function (Efe et al, 2005). Meanwhile, tbc-3 encodes a member of the highly conserved TBC (Tre-2, Bub2 and Cdc16) family of GTPase-activating proteins (GAPs) specific for Rab/Ypt GTPase (Lafourcade et al, 2004), and is expressed as two alternatively spliced forms, tbc-3a and tbc-3b according to the information in the C. elegans EST database (http://www.wormbase.org/) (Figure 4A and B). xh23 mutants harboured a missense mutation in the TBC domain (Asp234Asn) in the tbc-3 gene. This Asp residue is three residues next to the catalytic Arg237 and is completely conserved among the TBC family proteins (Figure 4C) (Pan et al, 2006). On the basis of molecular nature of the mutations and the fact that reduction of mon-2 and tbc-3 by RNAi resulted in suppressing the seam cell phenotype of ipla-1 mutants (Table I, see groups B and C), we conclude that suppression of ipla-1 seam cell defects is due to loss or strong reduction of function in these genes.
As mentioned above, ipla-1 functioned cell-autonomously in seam cells (Table I, see group A). To understand in which cells mon-2 and tbc-3 interact with ipla-1, we performed rescue experiments using the scm promoter in the ipla-1;sup backgrounds. Expression of mon-2 and tbc-3b fully rescued the suppression of the ipla-1 mutant phenotype by mon-2 and tbc-3 mutations, respectively (Table I, see group B and C, respectively). In contrast, no rescue was obtained by expression of tbc-3a in ipla-1;tbc-3 mutants. These results are consistent with our observation that tbc-3b but not tbc-3a is expressed in seam cells during the S4 divisions (Supplementary Figure 4). Taken together, all these results indicate that mon-2 and tbc-3 function cell-autonomously in seam cells for their interactions with ipla-1.
In yeast, the homologues of mon-2 (Mon2p) and tbc-3 (Gyp1p) are required for endosome-to-Golgi retrograde transport (Lafourcade et al, 2004; Efe et al, 2005; Gillingham et al, 2006). These previous reports led us to hypothesize that reducing the function of the retrograde transport pathway suppresses the seam cell phenotype of ipla-1 mutants. To test this possibility, we knocked down the expression of genes whose homologues are known to regulate endosome-to-Golgi retrograde transport in other species (Bonifacino and Rojas, 2006). We also reduced the expression of the genes whose homologues show physical or genetic interactions with Mon2p and Gyp1p in yeast (Lafourcade et al, 2004; Wicky et al, 2004; Efe et al, 2005; Gillingham et al, 2006).
We tested 50 genes listed in Supplementary Table 1 and identified seven genes that could suppress the seam cell phenotype of ipla-1 mutants. Among the newly identified suppressor genes listed in Table II, the functions of four genes have been previously well described: vps-26, vps-29, vps-35 and snx-1. The proteins encoded by these four genes form a large complex, termed the retromer complex, which is involved in retrograde transport of intracellular sorting receptors such as yeast Vps10p and mammalian mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN) (Seaman, 2005). snx-3 is a C. elegans homologue of yeast Grd19p, which is required for the sorting of the t-SNARE Pep12p and the aminopeptidase Ste13p from late endosomes to the TGN (Voos and Stevens, 1998; Hettema et al, 2003). tat-5 and pad-1 are C. elegans homologues of yeast Neo1p (a putative aminophospholipid translocase) and Dop1p (a member of the Dopey leucine zipper-like family), respectively, both of which interact physically with Mon2p (Wicky et al, 2004; Efe et al, 2005; Gillingham et al, 2006). We also knocked down the expression of genes involved in other transport pathways and found that ipla-1 mutant phenotype was not suppressed by reduction of such genes (Supplementary Table 1 and data not shown). Next, we investigated the cell autonomy of these suppressor genes by tissue-specific RNAi strategy using RNAi-spreading defective sid-1 mutants (see Supplementary data). We knocked down the expression of each suppressor gene (tat-5, pad-1 and vps-35) in seam cells as well as in hyp7 in a cell-specific manner, and found that, in addition to mon-2 and tbc-3, these three genes functioned cell-autonomously in seam cells (Table I, see group D). Finally, we studied the subcellular localization of WRM-1 in ipla-1;sup backgrounds (sup refers to any suppressor gene). Knockdown of each suppressor gene (mon-2, tbc-3 and snx-1) in ipla-1 mutants revealed that in all cases, WRM-1 localized properly to the anterior cortex of seam cells prior to the S4 divisions (Figure 5A–D). Taken together, we conclude that WRM-1 mislocalization phenotype in ipla-1 mutants depends on the presence of the endosome-to-Golgi retrograde trafficking genes in seam cells.
The assembly of Wnt signalling components at specific subcellular locations is an important aspect of asymmetric divisions in C. elegans. We described here a previously poorly characterized phospholipase, ipla-1, that regulates the subcellular localizations of β-catenin wrm-1 in the terminal asymmetric divisions of the stem cell-like epithelial seam cells (the S4 divisions). We also showed that ipla-1 genetically interacts with a group of genes involved in endosome-to-Golgi retrograde trafficking, including the retromer complex genes. Our results strongly support the notion that ipla-1 functions as a regulator of membrane trafficking in the polarization of β-catenin wrm-1 before asymmetric divisions.
We have shown that ipla-1 mutants were affected in cell-fate specification after the S4 divisions. The major cause of this phenotype appears to be misregulation of cortical polarity of β-catenin wrm-1 prior to the S4 divisions, which is proposed to be translated into asymmetry of nuclear TCF pop-1 level between two daughter cells (POP-1 asymmetry) (Mizumoto and Sawa, 2007b). Indeed, the POP-1 asymmetry was also disrupted in ipla-1 mutants.
We provided two lines of evidence indicating the involvement of endosome-to-Golgi retrograde trafficking in the formation and/or maintenance of cortical asymmetry of β-catenin wrm-1. First, cell polarity defects of ipla-1 mutants were suppressed by mutations of the two genes, Arf GEF-like mon-2 and Rab GAP tbc-3, both of which are reported to be involved in endosome-to-Golgi retrograde transport (Lafourcade et al, 2004; Efe et al, 2005; Gillingham et al, 2006). Although the yeast homologue of mon-2 is reported to function in the cytoplasm-to-vacuole transport pathway (the Cvt pathway) besides retrograde trafficking (Efe et al, 2005), ipla-1 seam cell defects were not suppressed by knockdown of other Cvt pathway genes (Supplementary Table 1, see group F). Second, knockdown of the retromer complex genes (vps-26, vps-29, vps-35 and snx-1) also resulted in suppression of WRM-1 mislocalization phenotype of ipla-1 mutants. The retromer is a coat-like complex that is required for efficient retrograde traffic from the endosome to the Golgi (Seaman, 2005). Cell-specific RNAi experiments revealed that the retrograde trafficking acts cell-autonomously in seam cells to modulate the asymmetric cortical localization of WRM-1.
The asymmetric distribution of proteins on distinct plasma membrane domains is a fundamental property of polarized epithelial cells and is formed in part by biosynthetic sorting from the Golgi (Mostov et al, 2003; Rodriguez-Boulan et al, 2005). In addition, a recent study has demonstrated that lysosomal degradation of endocytosed proteins is important for maintenance of cell polarity; a failure in endosomal entry and progression towards lysosomal degradation cause an expansion of the apical determinant proteins to basolateral surfaces in Drosophila epithelial cells (Lu and Bilder, 2005). Meanwhile, the endosome-to-Golgi retrograde transport pathway genes have roles in preventing lysosomal sorting and degradation of the cargo proteins (Arighi et al, 2004; Lafourcade et al, 2004; Seaman, 2004); in other words, the retrograde transport and the lysosomal degradation are alternative pathways, and thus anything that directly affects one is likely to affect the other indirectly. We showed in this study that expression of the WRM-1 mislocalization phenotype in ipla-1 mutants depends on the genes implicated in the retrograde transport pathway. In view of these results, we propose, as a possible explanation of our observations, that in wild-type seam cells ipla-1 promotes degradation of endocytosed proteins directly, or indirectly by inhibiting the retrograde transport pathway, to maintain the cortical asymmetry of β-catenin wrm-1 (schematized in Figure 5E). In ipla-1 mutants, endocytosed proteins may not be degraded, but alternatively transported to the Golgi, and then mistransported to various plasma membrane domains (Figure 5F). When sup genes are mutated or inactivated in ipla-1 mutants, such mistargeting may not occur and endocytosed proteins may be retained in the endosome or progress towards the lysosome, resulting in suppression of WRM-1 mislocalization phenotype (Figure 5G; sup refers to any suppressor gene listed in Table II). What are the proteins that are transported via an ipla-1-dependent mechanism in the S4 divisions? Possible candidates are the proteins that can mediate the cortical localization of β-catenin wrm-1. Interestingly, WRM-1::GFP is also localized as dots of fluorescence in the cytoplasm, which colocalizes with APC apr-1 and Axin pry-1 (Mizumoto and Sawa, 2007a), raising the possibility that vesicles containing these proteins are transported to the anterior cortex in mother cells. Other possible candidates are proteins that function upstream of β-catenin wrm-1, such as Dsh and Fz. Interestingly, Dsh dsh-2 can physically interact with subunits of the clathrin adaptor AP-1, which participates in membrane traffic between the endosome and the Golgi (Yu et al, 2007). Further experiments are needed to investigate our hypothesis, but this work is the first, to our knowledge, to raise the possibility that membrane-trafficking machinery can modulate the Wnt/β-catenin asymmetry pathway in a cell-autonomous manner.
We also found that ipla-1 mutants were defective in spindle orientation. It is believed that during asymmetric divisions, spindle positioning is governed by the interaction of astral microtubules with cortical factors that are localized asymmetrically in accordance with the polarity axis. In the asymmetric divisions of the EMS blastomere, spindle orientation is controlled by Fz mom-5 and Dsh dsh-2, but not by downstream nuclear effectors such as β-catenin wrm-1 and TCF pop-1 (Schlesinger et al, 1999; Walston et al, 2004). In the present study, we have shown that the asymmetry of cell fates was abolished in the S4 divisions that occurred properly along the A–P axis in ipla-1 mutants, which suggests that spindle orientation is controlled independently of the asymmetry of cortical β-catenin wrm-1 and nuclear TCF pop-1 in the S4 divisions. Given our results that spindle orientation phenotype of ipla-1 mutants was also almost completely suppressed by the mutations in both mon-2 and tbc-3, it is possible that a still unidentified cortical factor(s) which interacts with astral microtubules may be targeted by a similar mechanism to that of β-catenin wrm-1.
The retromer complex is reported to function non-cell-autonomously in Wnt-producing cells to control the polarity of the V5 seam cell division at the L1 stage, by creating a long-range gradient of Wnt egl-20 (Coudreuse et al, 2006). In addition, very recent studies have revealed that the retromer promotes Wnt secretion by effectively recycling Wntless mig-14, a putative cargo receptor for Wnt, from the endosome to the Golgi (Belenkaya et al, 2008; Franch-Marro et al, 2008; Pan et al, 2008; Port et al, 2008; Yang et al, 2008). As mentioned above, we have demonstrated that the retromer complex genes functioned cell-autonomously in seam cells for controlling the polarity of the S4 divisions. Moreover, ipla-1 seam cell defects were not suppressed by an egl-20 (n585) mutation or by RNAi of mig-14 (Supplementary Figure 5), suggesting that suppression of the ipla-1 mutant phenotypes is not due to reduced secretion of Wnt ligands. Thus, our findings show that the retromer complex not only functions in the Wnt-producing cells, but also in the Wnt-receiving cells.
The S4 divisions were specifically affected in ipla-1 mutants. We did not observe any defects in three well-studied asymmetric divisions: those of the EMS blastomere, the somatic gonadal precursor cells and the T-blast cell (Supplementary Figure 6 and data not shown). We currently have little experimental data that can explain this specificity; it is unlikely that ipla-1 is specifically expressed in seam cells at the timing of the S4 divisions as such stage-specific expression of IPLA-1 was not seen in our immunostaining experiments (Supplementary Figure 7 and Supplementary Movie 1); similarly to ipla-1, tbc-3 is not specifically expressed in seam cells just prior to the S4 divisions but expressed in seam cells in the earlier stages (Supplementary Figure 4). Furthermore, previous studies have demonstrated that the expression of vps-35, one of the suppressor genes for ipla-1 mutation, is not only limited to seam cells, but is also observed in body wall muscle, intestine, somatic gonad, pharynx and neuronal cells (Coudreuse et al, 2006; Prasad and Clark, 2006). During animal development, membrane traffic pathways are modified to accommodate the specific needs of individual cell types (Mostov et al, 2003). As seam cells in the S4 divisions are larger in size as compared with those in the asymmetric divisions at early larval stages, an additional mechanism provided by ipla-1 may be required for maintenance of the cortical polarity of β-catenin wrm-1.
In a genetic screen for shoot gravitropism mutants in Arabidopsis, a homologue of intracellular PLA1 (SGR2) and a SNARE protein (SGR4) were found to be important for gravity sensing (Kato et al, 2002). A mammalian intracellular PLA1 family member, p125 was previously shown to interact with the COPII component Sec23 (Tani et al, 1999). These studies, together with our findings, strongly suggest that ipla-1 acts as a regulator of membrane trafficking. A key emerging question is how a lipid-metabolizing enzyme, intracellular PLA1 controls membrane trafficking. It is notable that we identified tat-5, a putative aminophospholipid translocase, as a genetic suppressor of ipla-1 mutants. Aminophospholipid translocases, which specifically transport aminophospholipids such as phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the membrane, are believed to drive the formation of vesicles either by inducing the bending of membrane to facilitate vesicle budding, or by recruiting proteins for vesicle budding (Graham, 2004). It is tempting to speculate that ipla-1 may control endosome-to-Golgi retrograde transport by hydrolysing the aminophospholipids that are transported to the inner leaflet by the activity of tat-5. To support this idea, we have recently shown that aminophospholipids are preferred substrates for intracellular PLA1s (Nakajima et al, 2002; Morikawa et al, 2007).
Wnt signalling is implicated in the maintenance of stem cells that undergo asymmetric divisions (Aubert et al, 2002; Reya et al, 2003). Moreover, given ipla-1's striking evolutionary conservation, as well as the conservation of all the suppressor genes identified in this study, our results raise the possibility that intracellular PLA1 and the retrograde transport pathway genes control asymmetric divisions of stem cells in other animals.
Maintenance and genetic manipulation of C. elegans were carried out as described (Brenner, 1974). The following mutations and integrated transgenes were used: ipla-1(xh13, tm471)II, mon-2(xh22)IV, tbc-3(xh23)IV, sid-1(qt2)V, wIs51[scm::gfp], osIs10[scm::wrm-1::venus] and qIs74[gfp::pop-1]. Detailed information is included in the Supplementary data.
Click here for additional data file (emboj2008102s1.mov)
Click here for additional data file (emboj2008102s2.pdf)
We thank R Morikawa, K Mizumoto, DD Ikeda, Y Iino, M Fukuyama, K Kontani, H Kagoshima and M Miura for technical advice and encouragement; members of Sawa lab and Tsujimoto lab for helpful discussions and comments on this study; H Fukuda and Y Funakoshi for technical support and the Caenorhabditis Genetic Center (University of Minnesota, Minneapolis), A Fire, MJ Stern, J Audhya, HC Korswagen, WA Mohler and I Greenwald for strains and plasmids. TK was supported by the Junior Research Associate Program, RIKEN.
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Keywords: asymmetric divisions, C. elegans, phospholipase, retrograde trafficking, the Wnt/β-catenin asymmetry pathway.
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