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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.

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Related in: MedlinePlus

VAB-1 inhibits the UNC-34/NCK-1 complex and negatively regulates UNC-34 protein levels.(A) NCK-1 (GST) pull-down experiments. NCK-1 can pull-down UNC-34 (lane 3). Adding VAB-1 (either co-expressed (high VAB-1), lane 4, or mixing (low VAB-1), lane 5) inhibits the interaction between NCK-1 and UNC-34. VAB-1, NCK-1 and GST protein levels shown below, the dashed line indicates a cropped region from the blot. Tagged protein fusions used: GST-NCK-1, HIS-VAB-1, and MBP-UNC-34. Proteins were detected with antibodies to GST, MBP or VAB-1. (B) Inducing hyperactive MYR-VAB-1 (quIs16) via a heat shock promoter reduces the levels of UNC-34::GFP. unc-34::gfp transgenic animals had a GFP relative mean intensity of 1±0.07 (N = 12) under heat shock conditions, while hs:myr-vab-1;unc-34::gfp animals had a GFP relative mean intensity of 0.56±04 (p<0.01; student's t-test; N = 16) under the same conditions. All panels show UNC-34::GFP image of the CAN neuron. ‘N’ refers to the number of animals. (C) Images show PLM cells expressing mec-4::unc-34::gfp. In wild-type animals (left panel), the PLM shows high levels of UNC-34 expression and in contrast, the UNC-34 intensity is reduced in mry-vab-1 transgenic animals (right panel). Images were taken at identical exposure settings; dashed line outlines the PLM cell body; ‘N’ = number of animals; scale bar equals 2 µm.
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pgen-1002513-g005: VAB-1 inhibits the UNC-34/NCK-1 complex and negatively regulates UNC-34 protein levels.(A) NCK-1 (GST) pull-down experiments. NCK-1 can pull-down UNC-34 (lane 3). Adding VAB-1 (either co-expressed (high VAB-1), lane 4, or mixing (low VAB-1), lane 5) inhibits the interaction between NCK-1 and UNC-34. VAB-1, NCK-1 and GST protein levels shown below, the dashed line indicates a cropped region from the blot. Tagged protein fusions used: GST-NCK-1, HIS-VAB-1, and MBP-UNC-34. Proteins were detected with antibodies to GST, MBP or VAB-1. (B) Inducing hyperactive MYR-VAB-1 (quIs16) via a heat shock promoter reduces the levels of UNC-34::GFP. unc-34::gfp transgenic animals had a GFP relative mean intensity of 1±0.07 (N = 12) under heat shock conditions, while hs:myr-vab-1;unc-34::gfp animals had a GFP relative mean intensity of 0.56±04 (p<0.01; student's t-test; N = 16) under the same conditions. All panels show UNC-34::GFP image of the CAN neuron. ‘N’ refers to the number of animals. (C) Images show PLM cells expressing mec-4::unc-34::gfp. In wild-type animals (left panel), the PLM shows high levels of UNC-34 expression and in contrast, the UNC-34 intensity is reduced in mry-vab-1 transgenic animals (right panel). Images were taken at identical exposure settings; dashed line outlines the PLM cell body; ‘N’ = number of animals; scale bar equals 2 µm.

Mentions: Since UNC-34 and NCK-1 physically interact, we wanted to examine whether VAB-1, NCK-1 and UNC-34 could form a complex in vitro. Surprisingly, although UNC-34 can bind strongly to NCK-1, the introduction of VAB-1 abolished the binding between UNC-34 and NCK-1 (Figure 5A Lane 4, 5). This result suggests that VAB-1 might be inducing its effect at the growth cone membrane by relieving the inhibition of NCK-1 that is caused by UNC-34. To provide in vivo support of this we over expressed UNC-34 in the mechanosensory neurons (mec-4::unc-34) and it significantly reduced the MYR-VAB-1 PLM premature termination phenotype (Figure 1B).


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)

VAB-1 inhibits the UNC-34/NCK-1 complex and negatively regulates UNC-34 protein levels.(A) NCK-1 (GST) pull-down experiments. NCK-1 can pull-down UNC-34 (lane 3). Adding VAB-1 (either co-expressed (high VAB-1), lane 4, or mixing (low VAB-1), lane 5) inhibits the interaction between NCK-1 and UNC-34. VAB-1, NCK-1 and GST protein levels shown below, the dashed line indicates a cropped region from the blot. Tagged protein fusions used: GST-NCK-1, HIS-VAB-1, and MBP-UNC-34. Proteins were detected with antibodies to GST, MBP or VAB-1. (B) Inducing hyperactive MYR-VAB-1 (quIs16) via a heat shock promoter reduces the levels of UNC-34::GFP. unc-34::gfp transgenic animals had a GFP relative mean intensity of 1±0.07 (N = 12) under heat shock conditions, while hs:myr-vab-1;unc-34::gfp animals had a GFP relative mean intensity of 0.56±04 (p<0.01; student's t-test; N = 16) under the same conditions. All panels show UNC-34::GFP image of the CAN neuron. ‘N’ refers to the number of animals. (C) Images show PLM cells expressing mec-4::unc-34::gfp. In wild-type animals (left panel), the PLM shows high levels of UNC-34 expression and in contrast, the UNC-34 intensity is reduced in mry-vab-1 transgenic animals (right panel). Images were taken at identical exposure settings; dashed line outlines the PLM cell body; ‘N’ = number of animals; scale bar equals 2 µm.
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pgen-1002513-g005: VAB-1 inhibits the UNC-34/NCK-1 complex and negatively regulates UNC-34 protein levels.(A) NCK-1 (GST) pull-down experiments. NCK-1 can pull-down UNC-34 (lane 3). Adding VAB-1 (either co-expressed (high VAB-1), lane 4, or mixing (low VAB-1), lane 5) inhibits the interaction between NCK-1 and UNC-34. VAB-1, NCK-1 and GST protein levels shown below, the dashed line indicates a cropped region from the blot. Tagged protein fusions used: GST-NCK-1, HIS-VAB-1, and MBP-UNC-34. Proteins were detected with antibodies to GST, MBP or VAB-1. (B) Inducing hyperactive MYR-VAB-1 (quIs16) via a heat shock promoter reduces the levels of UNC-34::GFP. unc-34::gfp transgenic animals had a GFP relative mean intensity of 1±0.07 (N = 12) under heat shock conditions, while hs:myr-vab-1;unc-34::gfp animals had a GFP relative mean intensity of 0.56±04 (p<0.01; student's t-test; N = 16) under the same conditions. All panels show UNC-34::GFP image of the CAN neuron. ‘N’ refers to the number of animals. (C) Images show PLM cells expressing mec-4::unc-34::gfp. In wild-type animals (left panel), the PLM shows high levels of UNC-34 expression and in contrast, the UNC-34 intensity is reduced in mry-vab-1 transgenic animals (right panel). Images were taken at identical exposure settings; dashed line outlines the PLM cell body; ‘N’ = number of animals; scale bar equals 2 µm.
Mentions: Since UNC-34 and NCK-1 physically interact, we wanted to examine whether VAB-1, NCK-1 and UNC-34 could form a complex in vitro. Surprisingly, although UNC-34 can bind strongly to NCK-1, the introduction of VAB-1 abolished the binding between UNC-34 and NCK-1 (Figure 5A Lane 4, 5). This result suggests that VAB-1 might be inducing its effect at the growth cone membrane by relieving the inhibition of NCK-1 that is caused by UNC-34. To provide in vivo support of this we over expressed UNC-34 in the mechanosensory neurons (mec-4::unc-34) and it significantly reduced the MYR-VAB-1 PLM premature termination phenotype (Figure 1B).

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
Related in: MedlinePlus