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The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

Mohamed AM, Boudreau JR, Yu FP, Liu J, Chin-Sang ID - PLoS Genet. (2012)

Bottom Line: We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1.Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro.We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Queen's University, Kingston, Canada.

ABSTRACT
The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

Show MeSH
PLM defects in various backgrounds and their genetic interactions.(A) Young adults expressing mec-4::gfp(zdIs5). Anterior is to the left. Solid arrow points to where the PLM axon ends. A line diagram that corresponds to the morphology of the neuron is shown below. In wild-type animals (top gfp panel) the PLM axons terminate at the middle (vulva region triangle). MYR-VAB-1 (middle gfp panel) causes PLM axons to terminate before reaching their target. wsp-1(gm324) animals (bottom gfp panel) have PLM axons that overshoot past the vulva (triangle) and ALM neuron (dashed arrow). (B) The nck-1(ok694) and wsp-1(gm324) alleles significantly reduced the early termination defects caused by MYR-VAB-1. Over expressing unc-34 in the PLMs also reduced the MYR-VAB-1 termination defect. (C) vab-1, nck-1 and wsp-1 animals have PLM overextension defects. Reducing the levels of UNC-34 via tissue specific RNAi suppressed the PLM overextension defects of vab-1(dx31) and wsp-1(gm324). (D) unc-34(e566) loss-of-function and tissue specific unc-34 RNAi exhibit PLM axon termination. Over expression of NCK-1 in the mechanosensory neurons (mec-4::nck-1) caused low levels of the PLM early termination defects, but synergized in the unc-34(e566) background. Activating the Arp2/3 complex via the WSP-1 VCA domain (mec-4::vca) caused PLM axon termination defects. Error bars indicated the SEM, and significant differences between some of the strains were compared (using student's t-test), *P<0.05; **P<0.01; ***P<0.001; n.s. = not statistically significant. ‘N’ refers to the number of axons scored.
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pgen-1002513-g001: PLM defects in various backgrounds and their genetic interactions.(A) Young adults expressing mec-4::gfp(zdIs5). Anterior is to the left. Solid arrow points to where the PLM axon ends. A line diagram that corresponds to the morphology of the neuron is shown below. In wild-type animals (top gfp panel) the PLM axons terminate at the middle (vulva region triangle). MYR-VAB-1 (middle gfp panel) causes PLM axons to terminate before reaching their target. wsp-1(gm324) animals (bottom gfp panel) have PLM axons that overshoot past the vulva (triangle) and ALM neuron (dashed arrow). (B) The nck-1(ok694) and wsp-1(gm324) alleles significantly reduced the early termination defects caused by MYR-VAB-1. Over expressing unc-34 in the PLMs also reduced the MYR-VAB-1 termination defect. (C) vab-1, nck-1 and wsp-1 animals have PLM overextension defects. Reducing the levels of UNC-34 via tissue specific RNAi suppressed the PLM overextension defects of vab-1(dx31) and wsp-1(gm324). (D) unc-34(e566) loss-of-function and tissue specific unc-34 RNAi exhibit PLM axon termination. Over expression of NCK-1 in the mechanosensory neurons (mec-4::nck-1) caused low levels of the PLM early termination defects, but synergized in the unc-34(e566) background. Activating the Arp2/3 complex via the WSP-1 VCA domain (mec-4::vca) caused PLM axon termination defects. Error bars indicated the SEM, and significant differences between some of the strains were compared (using student's t-test), *P<0.05; **P<0.01; ***P<0.001; n.s. = not statistically significant. ‘N’ refers to the number of axons scored.

Mentions: To identify VAB-1 Eph RTK effectors, we utilized transgenic animals carrying mec-4::myr-vab-1 (quIs5) which encodes a constitutively active VAB-1 tyrosine kinase (myristoylated-VAB-1 termed MYR-VAB-1) in the mechanosensory neurons [6]. In wild-type young adults, PLM neuron cell bodies are located in the tail region and have axons that stop at the centre of the animal (Figure 1A). We previously showed that myr-vab-1 caused neuronal defects in the mechanosensory neurons, in particular the premature termination of PLM axons (Figure 1A, 1B) [6]. Since the MYR-VAB-1 behaves as a constitutively active VAB-1 RTK, we reasoned that mutations in effectors of the VAB-1 signal may suppress the neuronal defects. We used a candidate gene approach to examine genes with known roles in axon guidance and tested whether loss-of-function mutations could suppress the myr-vab-1 PLM premature termination phenotype. We identified nck-1 as a candidate effector of VAB-1 Eph RTK signaling. The nck-1(ok694) mutation partially suppressed the PLM axon premature termination (Figure 1B), indicating that other effectors are involved in the MYR-VAB-1 signaling. The C. elegans genome encodes for only one nck-1 adaptor protein, and is most similar to the human Nck2 and Drosophila DOCK [12]. NCK-1 has all the domain features of the NCK adaptor proteins, including three SH3 domains followed by a single SH2 domain. We previously reported that the deletion allele nck-1(ok694) is predicted to be a allele, thus all of our genetic work was carried out using the ok694 allele [12].


The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

Mohamed AM, Boudreau JR, Yu FP, Liu J, Chin-Sang ID - PLoS Genet. (2012)

PLM defects in various backgrounds and their genetic interactions.(A) Young adults expressing mec-4::gfp(zdIs5). Anterior is to the left. Solid arrow points to where the PLM axon ends. A line diagram that corresponds to the morphology of the neuron is shown below. In wild-type animals (top gfp panel) the PLM axons terminate at the middle (vulva region triangle). MYR-VAB-1 (middle gfp panel) causes PLM axons to terminate before reaching their target. wsp-1(gm324) animals (bottom gfp panel) have PLM axons that overshoot past the vulva (triangle) and ALM neuron (dashed arrow). (B) The nck-1(ok694) and wsp-1(gm324) alleles significantly reduced the early termination defects caused by MYR-VAB-1. Over expressing unc-34 in the PLMs also reduced the MYR-VAB-1 termination defect. (C) vab-1, nck-1 and wsp-1 animals have PLM overextension defects. Reducing the levels of UNC-34 via tissue specific RNAi suppressed the PLM overextension defects of vab-1(dx31) and wsp-1(gm324). (D) unc-34(e566) loss-of-function and tissue specific unc-34 RNAi exhibit PLM axon termination. Over expression of NCK-1 in the mechanosensory neurons (mec-4::nck-1) caused low levels of the PLM early termination defects, but synergized in the unc-34(e566) background. Activating the Arp2/3 complex via the WSP-1 VCA domain (mec-4::vca) caused PLM axon termination defects. Error bars indicated the SEM, and significant differences between some of the strains were compared (using student's t-test), *P<0.05; **P<0.01; ***P<0.001; n.s. = not statistically significant. ‘N’ refers to the number of axons scored.
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pgen-1002513-g001: PLM defects in various backgrounds and their genetic interactions.(A) Young adults expressing mec-4::gfp(zdIs5). Anterior is to the left. Solid arrow points to where the PLM axon ends. A line diagram that corresponds to the morphology of the neuron is shown below. In wild-type animals (top gfp panel) the PLM axons terminate at the middle (vulva region triangle). MYR-VAB-1 (middle gfp panel) causes PLM axons to terminate before reaching their target. wsp-1(gm324) animals (bottom gfp panel) have PLM axons that overshoot past the vulva (triangle) and ALM neuron (dashed arrow). (B) The nck-1(ok694) and wsp-1(gm324) alleles significantly reduced the early termination defects caused by MYR-VAB-1. Over expressing unc-34 in the PLMs also reduced the MYR-VAB-1 termination defect. (C) vab-1, nck-1 and wsp-1 animals have PLM overextension defects. Reducing the levels of UNC-34 via tissue specific RNAi suppressed the PLM overextension defects of vab-1(dx31) and wsp-1(gm324). (D) unc-34(e566) loss-of-function and tissue specific unc-34 RNAi exhibit PLM axon termination. Over expression of NCK-1 in the mechanosensory neurons (mec-4::nck-1) caused low levels of the PLM early termination defects, but synergized in the unc-34(e566) background. Activating the Arp2/3 complex via the WSP-1 VCA domain (mec-4::vca) caused PLM axon termination defects. Error bars indicated the SEM, and significant differences between some of the strains were compared (using student's t-test), *P<0.05; **P<0.01; ***P<0.001; n.s. = not statistically significant. ‘N’ refers to the number of axons scored.
Mentions: To identify VAB-1 Eph RTK effectors, we utilized transgenic animals carrying mec-4::myr-vab-1 (quIs5) which encodes a constitutively active VAB-1 tyrosine kinase (myristoylated-VAB-1 termed MYR-VAB-1) in the mechanosensory neurons [6]. In wild-type young adults, PLM neuron cell bodies are located in the tail region and have axons that stop at the centre of the animal (Figure 1A). We previously showed that myr-vab-1 caused neuronal defects in the mechanosensory neurons, in particular the premature termination of PLM axons (Figure 1A, 1B) [6]. Since the MYR-VAB-1 behaves as a constitutively active VAB-1 RTK, we reasoned that mutations in effectors of the VAB-1 signal may suppress the neuronal defects. We used a candidate gene approach to examine genes with known roles in axon guidance and tested whether loss-of-function mutations could suppress the myr-vab-1 PLM premature termination phenotype. We identified nck-1 as a candidate effector of VAB-1 Eph RTK signaling. The nck-1(ok694) mutation partially suppressed the PLM axon premature termination (Figure 1B), indicating that other effectors are involved in the MYR-VAB-1 signaling. The C. elegans genome encodes for only one nck-1 adaptor protein, and is most similar to the human Nck2 and Drosophila DOCK [12]. NCK-1 has all the domain features of the NCK adaptor proteins, including three SH3 domains followed by a single SH2 domain. We previously reported that the deletion allele nck-1(ok694) is predicted to be a allele, thus all of our genetic work was carried out using the ok694 allele [12].

Bottom Line: We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1.Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro.We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Queen's University, Kingston, Canada.

ABSTRACT
The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

Show MeSH