A DNA replication-independent function of pre-replication complex genes during cell invasion in C. elegans.

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.

Polarized epidermal growth factor secretion ensures robust vulval cell fate specification in Caenorhabditis elegans.

The anchor cell (AC) in C. elegans secretes an epidermal growth factor (EGF) homolog that induces adjacent vulval precursor cells (VPCs) to differentiate. The EGF receptor in the nearest VPC sequesters the limiting EGF amounts released by the AC to prevent EGF from spreading to distal VPCs. Here, we show that not only EGFR localization in the VPCs but also EGF polarity in the AC is necessary for robust fate specification. The AC secretes EGF in a directional manner towards the nearest VPC. Loss of AC polarity causes signal spreading and, when combined with MAPK pathway hyperactivation, the ectopic induction of distal VPCs. In a screen for genes preventing distal VPC induction, we identified sra-9 and nlp-26 as genes specifically required for polarized EGF secretion. sra-9(lf) and nlp-26(lf) mutants exhibit errors in vulval fate specification, reduced precision in VPC to AC alignment and increased variability in MAPK activation. sra-9 encodes a seven-pass transmembrane receptor acting in the AC and nlp-26 a neuropeptide-like protein expressed in the VPCs. SRA-9 and NLP-26 may transduce a feedback signal to channel EGF secretion towards the nearest VPC.

ß-Integrin de-phosphorylation by the Density-Enhanced Phosphatase DEP-1 attenuates EGFR signaling in C. elegans.

Density-Enhanced Phosphatase-1 (DEP-1) de-phosphorylates various growth factor receptors and adhesion proteins to regulate cell proliferation, adhesion and migration. Moreover, dep-1/scc1 mutations have been detected in various types of human cancers, indicating a broad tumor suppressor activity. During C. elegans development, DEP-1 mediates binary cell fate decisions by negatively regulating EGFR signaling. Using a substrate-trapping DEP-1 mutant in a proteomics approach, we have identified the C. elegans ß-integrin subunit PAT-3 as a specific DEP-1 substrate. DEP-1 selectively de-phosphorylates tyrosine 792 in the membrane-proximal NPXY motif to promote integrin activation via talin recruitment. The non-phosphorylatable ß-integrin mutant pat-3(Y792F) partially suppresses the hyperactive EGFR signaling phenotype caused by loss of dep-1 function. Thus, DEP-1 attenuates EGFR signaling in part by de-phosphorylating Y792 in the ß-integrin cytoplasmic tail, besides the direct de-phosphorylation of the EGFR. Furthermore, in vivo FRAP analysis indicates that the αß-integrin/talin complex attenuates EGFR signaling by restricting receptor mobility on the basolateral plasma membrane. We propose that DEP-1 regulates EGFR signaling via two parallel mechanisms, by direct receptor de-phosphorylation and by restricting receptor mobility through αß-integrin activation.

An in vivo EGF receptor localization screen in C. elegans Identifies the Ezrin homolog ERM-1 as a temporal regulator of signaling.

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.

PTEN negatively regulates MAPK signaling during Caenorhabditis elegans vulval development.

Vulval development in Caenorhabditis elegans serves as an excellent model to examine the crosstalk between different conserved signaling pathways that are deregulated in human cancer. The concerted action of the RAS/MAPK, NOTCH, and WNT pathways determines an invariant pattern of cell fates in three vulval precursor cells. We have discovered a novel form of crosstalk between components of the Insulin and the RAS/MAPK pathways. The insulin receptor DAF-2 stimulates, while DAF-18 PTEN inhibits, RAS/MAPK signaling in the vulval precursor cells. Surprisingly, the inhibitory activity of DAF-18 PTEN on the RAS/MAPK pathway is partially independent of its PIP(3) lipid phosphatase activity and does not involve further downstream components of the insulin pathway, such as AKT and DAF-16 FOXO. Genetic and biochemical analyses indicate that DAF-18 negatively regulates vulval induction by inhibiting MAPK activation. Thus, mutations in the PTEN tumor suppressor gene may result in the simultaneous hyper-activation of two oncogenic signaling pathways.

The CHORD protein CHP-1 regulates EGF receptor trafficking and signaling in C. elegans and in human cells.

The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. Here, we show that the putative HSP-90 co-chaperone CHP-1 acts as a regulator of EGFR trafficking in C. elegans. Loss of chp-1 causes the retention of the EGFR in the ER and decreases MAPK signaling. CHP-1 is specifically required for EGFR trafficking, as the localization of other transmembrane receptors is unaltered in chp-1(lf) mutants, and the inhibition of hsp-90 or other co-chaperones does not affect EGFR localization. The role of the CHP-1 homolog CHORDC1 during EGFR trafficking is conserved in human cells. Analogous to C. elegans, the response of CHORDC1-deficient A431 cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity decreases. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate that CHP-1 plays an HSP90-independent function in controlling EGFR trafficking through the ER.

A conserved function of C. elegans CASY-1 calsyntenin in associative learning.

BACKGROUND: Whole-genome association studies in humans have enabled the unbiased discovery of new genes associated with human memory performance. However, such studies do not allow for a functional or causal testing of newly identified candidate genes. Since polymorphisms in Calsyntenin 2 (CLSTN2) showed a significant association with episodic memory performance in humans, we tested the C. elegans CLSTN2 ortholog CASY-1 for possible functions in the associative behavior of C. elegans. METHODOLOGY/PRINCIPAL FINDINGS: Using three different associative learning paradigms and functional rescue experiments, we show that CASY-1 plays an important role during associative learning in C. elegans. Furthermore, neuronal expression of human CLSTN2 in C. elegans rescues the learning defects of casy-1 mutants. Finally, genetic interaction studies and neuron-specific expression experiments suggest that CASY-1 may regulate AMPA-like GLR-1 glutamate receptor signaling. CONCLUSION/SIGNIFICANCE: Our experiments demonstrate a remarkable conservation of the molecular function of Calsyntenins between nematodes and humans and point at a role of C. elegans casy-1 in regulating a glutamate receptor signaling pathway.