LIN-12/Notch trafficking and regulation of DSL ligand activity during vulval induction in Caenorhabditis elegans

A novel mode of crosstalk between the EGFR-Ras-MAPK and LIN-12/Notch pathways occurs during the patterning of a row of vulval precursor cells (VPCs) in Caenorhabditis elegans: activation of the EGFR-Ras-MAPK pathway in the central VPC promotes endocytosis and degradation of LIN-12 protein. LIN-12 downregulation in the central VPC is a prerequisite for the activity of the lateral signal, which activates LIN-12 in neighboring VPCs. Here we characterize cis-acting targeting sequences in the LIN-12 intracellular domain and find that in addition to a di-leucine motif, serine/threonine residues are important for internalization and lysine residues are important for post-internalization trafficking and degradation. We also identify two trans-acting factors that are required for post-internalization trafficking and degradation: ALX-1, a homolog of yeast Bro1p and mammalian Alix and the WWP-1/Su(dx)/Itch ubiquitin ligase. By examining the effects of mutated forms of LIN-12 and reduced wwp-1 or alx-1 activity on subcellular localization and activity of LIN-12, we provide evidence that the lateral signal-inhibiting activity of LIN-12 resides in the extracellular domain and occurs at the apical surface of the VPCs.     

An AGEF-1/Arf GTPase/AP-1 Ensemble Antagonizes LET-23 EGFR Basolateral Localization and Signaling during C. elegans Vulva Induction

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.     

p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans.

Mutations in the Caenorhabditis elegans sel-9 gene elevate the activity of lin-12 and glp-1, which encode members of the LIN-12/NOTCH family of receptors. Sequence analysis indicates SEL-9 is one of several C. elegans p24 proteins. Allele-specific genetic interactions suggest that reducing sel-9 activity increases the activity of mutations altering the extracellular domains of LIN-12 or GLP-1. Reducing sel-9 activity restores the trafficking to the plasma membrane of a mutant GLP-1 protein that would otherwise accumulate within the cell. Our results suggest a role for SEL-9 and other p24 proteins in the negative regulation of transport of LIN-12 and GLP-1 to the cell surface, and favor a role for p24 proteins in a quality control mechanism for endoplasmic reticulum-Golgi transport.     

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.     

sel-7, a positive regulator of lin-12 activity, encodes a novel nuclear protein in Caenorhabditis elegans

Suppressor genetics in C. elegans has identified key components of the LIN-12/Notch signaling pathway. Here, we describe a genetic and molecular characterization of the suppressor gene sel-7. We show that reducing or eliminating sel-7 activity suppresses the effects of constitutive lin-12 activity, enhances the effects of partially reduced lin-12 activity, and causes a synthetic Lin-12(0) phenotype when combined with a null mutation in the sel-12 presenilin gene. These observations suggest that sel-7 is a positive regulator of lin-12 activity. We also show that SEL-7 encodes a novel nuclear protein. Through yeast two-hybrid screening, we identified an apparent interaction partner, K08E3.8, that also interacts with SEL-8, a known component of the nuclear complex that forms upon LIN-12 activation. Our data suggest potential roles for SEL-7 in the assembly or function of this nuclear complex.     

APH-2/Nicastrin Functions in LIN-12/Notch Signaling in the Caenorhabditis elegans Somatic Gonad

Nicastrin is a recently identified member of high-molecular weight complexes containing presenilin. The Caenorhabditis elegans homolog of nicastrin, aph-2, was shown to be required for GLP-1/Notch signaling in the early embryo. In addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. We show that this latter defect is related to the SEL-12/presenilin egg-laying defect. We also show that aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. We illustrate a new role for aph-2 in facilitating lin-12 signaling in the somatic gonad, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Finally, we demonstrate that nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins.     

The Caenorhabditis Elegans Sel-1 Gene, a Negative Regulator of Lin-12 and Glp-1, Encodes a Predicted Extracellular Protein

The Caenorhabditis elegans lin-12 and glp-1 genes encode members of the LIN-12/NOTCH family of receptors. The sel-1 gene was identified as an extragenic suppressor of a lin-12 hypomorphic mutant. We show in this report that the sel-1 null phenotype is wild type, except for an apparent elevation in lin-12 and glp-1 activity in sensitized genetic backgrounds, and that this genetic interaction seems to be lin-12 and glp-1 specific. We also find that sel-1 encodes a predicted extracellular protein, with a domain sharing sequence similarity to predicted proteins from humans and yeast. SEL-1 may interact with the LIN-12 and GLP-1 receptors and/or their respective ligands to down-regulate signaling.     

Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.     

LIN-14 inhibition of LIN-12 contributes to precision and timing of C. elegans vulval fate patterning

Studies of C. elegans vulval development have illuminated mechanisms underlying cell fate specification and elucidated intercellular signaling pathways [1]. The vulval precursor cells (VPCs) are spatially patterned during the L3 stage by the EGFR-Ras-MAPK-mediated inductive signal and the LIN-12/Notch-mediated lateral signal. The pattern is both precise and robust [2] because of crosstalk between these pathways [3]. Signaling is also regulated temporally, because constitutive activation of the spatial patterning pathways does not alter the timing of VPC fate specification [4, 5]. The heterochronic genes, including the microRNA lin-4 and its target lin-14, constitute a temporal control mechanism used in different contexts [6-8]. We find that lin-4 specifically controls the activity of LIN-12/Notch through lin-14, but not other known targets, and that persistent lin-14 blocks LIN-12 activity without interfering with the key events of LIN-12/Notch signal transduction. In the L2 stage, there is sufficient lin-14 activity to inhibit constitutive lin-12. Our results suggest that lin-4 and lin-14 contribute to spatial patterning through temporal gating of LIN-12. We propose that in the L2 stage, lin-14 sets a high threshold for LIN-12 activation to help prevent premature activation of LIN-12 by ligands expressed in other cells in the vicinity, thereby contributing to the precision and robustness of VPC fate patterning.     

SEL-5, a serine/threonine kinase that facilitates lin-12 activity in Caenorhabditis elegans

Ligands present on neighboring cells activate receptors of the LIN-12/Notch family by inducing a proteolytic cleavage event that releases the intracellular domain. Mutations that appear to eliminate sel-5 activity are able to suppress constitutive activity of lin-12(d) mutations that are point mutations in the extracellular domain of LIN-12, but cannot suppress lin-12(intra), the untethered intracellular domain. These results suggest that sel-5 acts prior to or during ligand-dependent release of the intracellular domain. In addition, sel-5 suppression of lin-12(d) mutations is tissue specific: loss of sel-5 activity can suppress defects in the anchor cell/ventral uterine precursor cell fate decision and a sex myoblast/coelomocyte decision, but cannot suppress defects in two different ventral hypodermal cell fate decisions in hermaphrodites and males. sel-5 encodes at least two proteins, from alternatively spliced mRNAs, that share an amino-terminal region and differ in the carboxy-terminal region. The amino-terminal region contains the hallmarks of a serine/threonine kinase domain, which is most similar to mammalian GAK1 and yeast Pak1p.     

Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development

By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. We have identified EGFR substrate protein-8 (EPS-8) as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis.     

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.     

Competence and commitment of Caenorhabditis elegans vulval precursor cells.

Multipotent Caenorhabditis elegans vulval precursor cells (VPCs) choose among three fates (1 degrees, 2 degrees, and 3 degrees ) in response to two intercellular signals: the EGF family growth factor LIN-3 induces 1 degrees fates at high levels and 2 degrees fates at low levels; and a signal via the receptor LIN-12 induces 2 degrees fates. If the level of LIN-3 signal is reduced by a lin-3 hypomorphic mutation, the daughters of the VPC closest to the anchor cell (AC), P6.p, are induced by the AC. By expressing LIN-3 as a function of time in LIN-3-deficient animals, we find that both VPCs and the daughters of VPCs are competent to respond to LIN-3, and VPC daughters lose competence after fusing with the hypodermis. We also demonstrate that the daughters of VPCs specified to be 2 degrees can respond to LIN-3, indicating that 2 degrees VPCs are not irreversibly committed. We propose that maintenance of VPC competence after the first cell cycle and the prioritization of the 1 degrees fate help ensure that P6.p will become 1 degrees. This mechanism of competence regulation might have been maintained from ancestral nematode species that used induction both before and after VPC division and serves to maximize the probability that a functional vulva is formed.     

RAB-7 antagonizes LET-23 EGFR signaling during vulva development in Caenorhabditis elegans

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.     

A loss of function mutant of the presenilin homologue SEL-12 undergoes aberrant endoproteolysis in Caenorhabditis elegans and increases abeta 42 generation in human cells

The familial Alzheimer's disease-associated presenilins (PSs) occur as a dimeric complex of proteolytically generated fragments, which functionally supports endoproteolysis of Notch and the beta-amyloid precursor protein (betaAPP). A homologous gene, sel-12, has been identified in Caenorhabditis elegans. We now demonstrate that wild-type (wt) SEL-12 undergoes endoproteolytic cleavage in C. elegans similar to the PSs in human tissue. In contrast, SEL-12 C60S protein expressed from the sel-12(ar131) allele is miscleaved in C. elegans, resulting in a larger mutant N-terminal fragment. Neither SEL-12 wt nor C60S undergo endoproteolytic processing upon expression in human cells, suggesting that SEL-12 is cleaved by a C. elegans-specific endoproteolytic activity. The loss of function of sel-12 in C. elegans is not associated with a dominant negative activity in human cells, because SEL-12 C60S and the corresponding PS1 C92S mutation do not interfere with Notch1 cleavage. Moreover, both mutant variants increase the aberrant production of the highly amyloidogenic 42-amino acid version of amyloid beta-peptide similar to familial Alzheimer's disease-associated human PS mutants. Our data therefore demonstrate that the C60S mutation in SEL-12 is associated with aberrant endoproteolysis and a loss of function in C. elegans, whereas a gain of misfunction is observed upon expression in human cells.     

Wnt and EGF pathways act together to induce C. elegans male hook development

Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1°, 2°, 3°), and 2° HCG fate specification is mediated by LIN-12/Notch. We show that 2° HCG specification depends on the presence of a cell with the 1° fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1° and 2° HCG fate. A requirement for EGF signaling during 1° fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1° lineage, and LIN-12/NOTCH induces a 2° lineage. Wnt signaling is also required for execution of the 1° and 2° HCG lineages. lin-17 and bar-1/β-catenin are preferentially expressed in the presumptive 1° cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.