Different Mi-2 complexes for various developmental functions in Caenorhabditis elegans

Biochemical purifications from mammalian cells and Xenopus oocytes revealed that vertebrate Mi-2 proteins reside in multisubunit NuRD (Nucleosome Remodeling and Deacetylase) complexes. Since all NuRD subunits are highly conserved in the genomes of C. elegans and Drosophila, it was suggested that NuRD complexes also exist in invertebrates. Recently, a novel dMec complex, composed of dMi-2 and dMEP-1 was identified in Drosophila. The genome of C. elegans encodes two highly homologous Mi-2 orthologues, LET-418 and CHD-3. Here we demonstrate that these proteins define at least three different protein complexes, two distinct NuRD complexes and one MEC complex. The two canonical NuRD complexes share the same core subunits HDA-1/HDAC, LIN-53/RbAp and LIN-40/MTA, but differ in their Mi-2 orthologues LET-418 or CHD-3. LET-418 but not CHD-3, interacts with the Krüppel-like protein MEP-1 in a distinct complex, the MEC complex. Based on microarrays analyses, we propose that MEC constitutes an important LET-418 containing regulatory complex during C. elegans embryonic and early larval development. It is required for the repression of germline potential in somatic cells and acts when blastomeres are still dividing and differentiating. The two NuRD complexes may not be important for the early development, but may act later during postembryonic development. Altogether, our data suggest a considerable complexity in the composition, the developmental function and the tissue-specificity of the different C. elegans Mi-2 complexes.     

Asymmetric segregation of PIE-1 in C. elegans is mediated by two complementary mechanisms that act through separate PIE-1 protein domains

The CCCH finger protein PIE-1 is a regulator of germ cell fate that segregates with the germ lineage in early embryos. At each asymmetric division, PIE-1 is inherited preferentially by the germline daughter and is excluded from the somatic daughter. We show that this asymmetry is regulated at the protein level by two complementary mechanisms. The first acts before cell division to enrich PIE-1 in the cytoplasm destined for the germline daughter. The second acts after cell division to eliminate any PIE-1 left in the somatic daughter. The latter mechanism depends on PIE-1's first CCCH finger (ZF1), which targets PIE-1 for degradation in somatic blastomeres. ZF1s in two other germline proteins, POS-1 and MEX-1, are also degraded in somatic blastomeres, suggesting that localized degradation also acts on these proteins to exclude them from somatic lineages.     

The C. elegans sex determination protein MOG-3 functions in meiosis and binds to the CSL co-repressor CIR-1

In the germ line of the Caenorhabditis elegans hermaphrodite, nuclei either proliferate through mitosis or initiate meiosis, finally differentiating as spermatids or oocytes. The production of oocytes requires repression of the fem-3 mRNA by cytoplasmic FBF and nuclear MOG proteins. Here we report the identification of the sex determining gene mog-3 and show that in addition to its role in gamete sex determination, it is necessary for meiosis by acting downstream of GLP-1/Notch. Furthermore, we found that MOG-3 binds both to the nuclear proteins MEP-1 and CIR-1. MEP-1 is necessary for oocyte production and somatic differentiation, while the mammalian CIR-1 homolog counters Notch signaling. We propose that MOG-3, MEP-1 and CIR-1 associate in a nuclear complex which regulates different aspects of germ cell development. While FBF triggers the sperm/oocyte switch by directly repressing the fem-3 mRNA in the cytoplasm, the MOG proteins play a more indirect role in the nucleus, perhaps by acting as epigenetic regulators or by controlling precise splicing events.     

SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells

The vulval precursor cells (VPCs) of Caenorhabditis elegans are polarized epithelial cells that adopt a precise pattern of fates through regulated activity of basolateral LET-23/EGF receptor and apical LIN-12/Notch. During VPC patterning, there is reciprocal modulation of endocytosis and trafficking of both LET-23 and LIN-12. We identified sel-2 as a negative regulator of lin-12/Notch activity in the VPCs, and found that SEL-2 is the homolog of two closely related human proteins, neurobeachin (also known as BCL8B) and LPS-responsive, beige-like anchor protein (LRBA). SEL-2, neurobeachin and LRBA belong to a distinct subfamily of BEACH-WD40 domain-containing proteins. Loss of sel-2 activity leads to basolateral mislocalization and increased accumulation of LIN-12 in VPCs in which LET-23 is not active, and to impaired downregulation of basolateral LET-23 in VPCs in which LIN-12 is active. Downregulation of apical LIN-12 in the VPC in which LET-23 is active is not affected. In addition, in sel-2 mutants, the polarized cells of the intestinal epithelium display an aberrant accumulation of the lipophilic dye FM4-64 when the dye is presented to the basolateral surface. Our observations indicate that SEL-2/neurobeachin/LRBA is involved in endosomal traffic and may be involved in efficient delivery of cell surface proteins to the lysosome. Our results also suggest that sel-2 activity may contribute to the appropriate steady-state level of LIN-12 or to trafficking events that affect receptor activation.     

LIN-10 can promote LET-23 EGFR signaling and trafficking independently of LIN-2 and LIN-7

During Caenorhabditis elegans larval development, an inductive signal mediated by the LET-23 EGFR (epidermal growth factor receptor), specifies three of six vulva precursor cells (VPCs) to adopt vulval cell fates. An evolutionarily conserved complex consisting of PDZ domain-containing scaffold proteins LIN-2 (CASK), LIN-7 (Lin7 or Veli), and LIN-10 (APBA1 or Mint1) (LIN-2/7/10) mediates basolateral LET-23 EGFR localization in the VPCs to permit signal transmission and development of the vulva. We recently found that the LIN-2/7/10 complex likely forms at Golgi ministacks; however, the mechanism through which the complex targets the receptor to the basolateral membrane remains unknown. Here we found that overexpression of LIN-10 or LIN-7 can compensate for loss of their complex components by promoting LET-23 EGFR signaling through previously unknown complex-independent and receptor-dependent pathways. In particular, LIN-10 can independently promote basolateral LET-23 EGFR localization, and its complex-independent function uniquely requires its PDZ domains that also regulate its localization to Golgi. These studies point to a novel complex-independent function for LIN-7 and LIN-10 that broadens our understanding of how this complex regulates targeted sorting of membrane proteins.     

Coupling between cytoplasmic concentration gradients through local control of protein mobility in the Caenorhabditis elegans zygote

Cell polarity is characterized by the asymmetric distribution of factors at the cell cortex and in the cytoplasm. Although mechanisms that establish cortical asymmetries have been characterized, less is known about how persistent cytoplasmic asymmetries are generated. During the asymmetric division of the Caenorhabditis elegans zygote, the PAR proteins orchestrate the segregation of the cytoplasmic RNA-binding proteins MEX-5/6 to the anterior cytoplasm and PIE-1, POS-1, and MEX-1 to the posterior cytoplasm. In this study, we find that MEX-5/6 control the segregation of GFP::PIE-1, GFP::POS-1, and GFP::MEX-1 by locally increasing their mobility in the anterior cytoplasm. Remarkably, PIE-1, POS-1, and MEX-1 form gradients with distinct strengths, which correlates with differences in their responsiveness to MEX-5/6. We show that MEX-5/6 act downstream of the polarity regulators PAR-1 and PAR-3 and in a concentration-dependent manner to increase the mobility of GFP::PIE-1. These findings suggest that the MEX-5/6 concentration gradients are directly coupled to the establishment of posterior-rich PIE-1, POS-1, and MEX-1 concentration gradients via the formation of anterior-fast, posterior-slow mobility gradients.     

LET-23-mediated signal transduction during Caenorhabditis elegans development

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming α-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the “vulvaless” phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the “multivulva” phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development—larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways. © 1995 wiley-Liss, Inc.     

Early determination in the C. elegans embryo: a gene, cib-1, required to specify a set of stem-cell-like blastomeres

The early somatic blastomeres founding the tissues in the C. elegans embryo are derived in a stem-cell-like lineage from the P cells. We have isolated maternal effect lethal mutations defining the gene cib-1 in which the P cells, P1-P3, skip a cell cycle and acquire the fates of only their somatic daughters. Therefore, the cib-1 gene is required for the specification of the stem-cell-like fate of these cells. The analysis of the development of these mutants suggests that the clock controlling the cell cycles in the early embryo is directly coupled to the fate of a cell and that there must be another developmental clock that activates the determinative inventory for the early decision-making.     

Functional phylogeny relates LET-756 to fibroblast growth factor 9

Fibroblast growth factors (FGFs) are secreted regulatory proteins involved in various developmental processes. In vertebrates, the FGF superfamily comprises 22 members. In non-vertebrates, six FGF genes have been identified in Ciona intestinalis, three in Drosophila melanogaster, and two (let-756 and egl-17) in Caenorhabditis elegans. The core of LET-756 shares a 30-50% sequence identity with the various members of the superfamily. The relationships between vertebrate and non-vertebrate FGFs are not clear. We made chimeric FGFs by replacing the core region of LET-756 by the cores of various mammalian, fly, and worm FGFs. LET-756 deleted in its core region was no longer able to rescue the lethal phenotype of a let-756 null mutant, and only chimeras containing the cores of FGFs 9, 16, and 20 showed rescue capacity. This core contains an internal motif of six amino acid residues (EFISIA) whose deletion or mutation abolished both the rescue activity and FGF secretion in the supernatant of transfected COS-1 cells. Chimera containing the core of C. intestinalis FGF9/16/20, a potential ortholog of FGF9 lacking the complete EFISIA motif, was not able to rescue the lethal phenotype or be secreted. However, the introduction of the EFISIA motif restored both activities. The data show that the EFISIA motif in the core of LET-756 is essential for its biological activity and that FGFs 9, 16, and 20, which contain that motif, are functionally close to LET-756 and may be evolutionary related. This non-classical mode of secretion using an internal motif is conserved throughout evolution.